CN110199881A - A kind of sweet potato health seedling cultural method removing sweet potato viruses complex SPVD - Google Patents
A kind of sweet potato health seedling cultural method removing sweet potato viruses complex SPVD Download PDFInfo
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- CN110199881A CN110199881A CN201910618618.7A CN201910618618A CN110199881A CN 110199881 A CN110199881 A CN 110199881A CN 201910618618 A CN201910618618 A CN 201910618618A CN 110199881 A CN110199881 A CN 110199881A
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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
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
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Abstract
The invention discloses a kind of sweet potato health seedling cultural methods for removing sweet potato viruses complex SPVD, belong to sweet potato technical field of tissue culture.It chooses sweet potato viruses complex SPVD and infects sweet potato sprout, the top stem section of clip band, it is soaked in after cultivating 96h in the 800 times of dilutions of phonetic peptide of Ningnan, take 3cm young tender stem tip, tap water rinses, Tween-80 is added to clean, again after rinsing with ruinning water, it is shaken and is sterilized with 0.1% sodium hypochlorite, then sterile purified water rinses, cut 1-2 phyllopodium shoot apical meristem of band, it is incubated at induction seedling culture medium, after cultivating seedling, selection regeneration plant is removed shoot apical meristem again and is cultivated, culture medium is still induction seedling culture medium, the sweet potato healthy plant for cultivating seedling is detected through RT-PCR and ELISA, sweet potato viruses complex SPVD removal efficiency is up to 100%, this method is a kind of method that sweet potato health seedling is cultivated in simple and effective SPVD removing.
Description
Technical field
The present invention relates to a kind of sweet potato health seedling cultural methods, and in particular to a kind of removing sweet potato viruses complex SPVD
Sweet potato health seedling culture method, belong to sweet potato cell engineering field.
Background technique
Sweet potato viruses complex SPVD (Sweetpotato virus disease) is by sweet potato chlorotic stunt virus
(Sweetpotato chlorotic stunt virus, SPCSV) and Sweet Potato Feathery Mottle Virus (Sweetpotato
Feathery mottle virus, SPFMV) assist life to infect disease caused by sweet potato altogether.The sweet potato performance plant for infecting SPVD is short
Changing, blade chlorisis, the symptoms such as deformity, SPVD influences yield of sweet potato greatly, 90% or more production loss can be caused when serious,
It even has no harvest, is the destructive disease of sweet potato.Currently, being mainly distributed on the ground such as Guangdong, Jiangsu, Sichuan, Anhui, Fujian, have after
Continuous diffusion tendency.Currently, having passed through a large amount of kinds that save identification, that production is featured occurs susceptible phenomenon, it there is no and found
Imitate antigen.Previous virus-free culture operation cannot effectively remove SPVD, and the detoxic seedling of cultivation SPVD removal efficiency after detecting is lower.
Summary of the invention
The present invention effectively compensates for the deficiency of former virus-free culture method, and it is multiple to provide a kind of effectively removing sweet potato viruses
The health seedling cultural method of fit SPVD, this method is easy to operate, practical, reproducible, and it is multiple can effectively to remove sweet potato viruses
Fit SPVD turns out the health seedling that sweet potato production promotes and applies.
The present invention is realized with following technical solution: a kind of sweet potato health seedling removing sweet potato viruses complex SPVD
Cultural method, it is characterised in that: choose infection SPVD virus plant and then lead to through the phonetic peptide immersion treatment in viral inhibitors Ningnan
Stem-apex Meristem culture is crossed, carries out stem-apex Meristem culture using regenerated plant again, it is multiple to cultivate removing sweet potato viruses
The sweet potato health seedling that fit SPVD infects；Concrete operations sequentially include the following steps:
(1) configuration of the phonetic 800 times of dilutions of peptide in Ningnan: taking the total active constituent content of 2.5 ml is 68% viral inhibitors Ningnan
Phonetic peptide solution adds Hongland nutrient solution to be settled to 2 L spare；
(2) clip sweet potato viruses complex SPVD infects 25 cm of sweet potato sprout band stem apex stem section, is incubated at the phonetic peptide in above-mentioned Ningnan
In 800 times of dilutions, 96 h of incubated at room temperature；
(3) stem-apex Meristem culture: the 3 cm young tender stem tips in Ningnan 800 times of dilution cultures of phonetic peptide are taken, are rushed through tap water
It washes 5-10 minutes, Tween-80 is added to clean 2-3 min, after then rinsing 2-3 min with tap water again, then with 0.1% hypochlorous acid
Sodium concussion 20 min of sterilizing, then rinsed 3-4 times with sterile purified water, it is mitogenetic that 1-2 phyllopodium stem apex of band is cut under anatomical lens
Tissue is incubated at induction seedling culture medium and obtains first time stem-apex Meristem culture regeneration plant after cultivating seedling, chooses
The regeneration plant carries out removing stem-apex Meristem culture again, and culture medium is still above-mentioned induction seedling culture medium, obtains second
Secondary stem-apex Meristem culture regeneration plant；
(4) sweet potato viruses complex SPVD is detected: the sweet potato health for choosing second of stem-apex Meristem culture regeneration plant is planted
Strain, carries out SPVD to it using RT-PCR and ELISA and infects detection；
The induction seedling culture medium refers to adds 6-BA, 1 mg/L in MS minimal medium, NAA, 0.01 mg/L, TDZ,
0.5 mg/L, pH value 5.8.
The invention has the advantages that this method is easy to operate, and it is practical, reproducible, there is the section for being engaged in tissue cultures basis
Grind, impart knowledge to students and enterprise can use this method fast culture removing sweet potato viruses complex SPVD sweet potato health seedling, be
A kind of effective ways of sweet potato health seedling culture.
Detailed description of the invention
Fig. 1 is influence of the different virus inhibitor processing to sweet potato stem tip separate living tissue planting percent；
Fig. 2 is Xu-shu No.22 and primary, the secondary stem-apex Meristem culture seedling viral diagnosis result of abundant Xu's potato 20；
Fig. 3 is SPFMV removal efficiency after the processing of different virus inhibitor.
Specific embodiment
Method and effect of the invention are further illustrated below with reference to embodiment, but are not limited the scope of the invention.
Embodiment 1
1. test material and method
1.1, test material: test selected materials are to detect through ELISA, RT-PCR and infect two of classical symptom with SPVD
Susceptible material Xu-shu No.22 and abundant Xu's potato 20, cutting propagation is spare under the conditions of isolation cover net.
, test method:
Viral inhibitors processing: this experiment selects viral inhibitors to have the phonetic peptide of lentinan, Ningnan, toxic fluoride phosphate, amino-oligosacchride
Element, the minimum concentration of respective suitable treatment is configured according to specification, and dilution selects Hongland solution, respectively 3 ml mushrooms
Hongland solution is added in polysaccharide, the phonetic peptide in 2.5 Ningnans ml, 3.4 g toxic fluoride phosphates, 3.4 ml amino-oligosaccharides, and constant volume 2L is standby
With.Choose Xu-shu No.22 and the susceptible material of abundant Xu's potato 20,96 h of incubated at room temperature in the above-mentioned HIV suppression agent solution prepared；20
Strain/kind/every viral inhibitors, is repeated 3 times；
Stem-apex Meristem culture: it takes in 3 processed cm of each viral inhibitors or so young tender stem tip, is rinsed through tap water
5-10 minutes, Tween-80 is added to clean 2-3 min, after then rinsing 2-3 min with tap water again, then with 0.1% sodium hypochlorite
Concussion 20 min of sterilizing, then rinsed 3-4 times with sterile purified water, 1-2 mitogenetic group of phyllopodium stem apex of band is cut under anatomical lens
It knits, is incubated at induction seedling culture medium, after cultivating seedling, obtain first time stem-apex Meristem culture regeneration plant, choosing should
Regeneration plant carries out removing stem-apex Meristem culture again, and culture medium is still above-mentioned induction seedling culture medium, obtains second
Stem-apex Meristem culture regeneration plant；
Sweet potato viruses complex SPVD detection: first time and its corresponding second of stem-apex Meristem culture regeneration plant are chosen
The sweet potato healthy plant of (seedling) carries out SPFMV and SPCSV to it using RT-PCR and ELISA and infects detection.
The induction seedling culture medium refers to addition 6-BA (6-benzyl aminopurine), 1 mg/L in MS minimal medium,
NAA(methyl α-naphthyl acetate), 0.01 mg/L, TDZ (Thidiazuron), 0.5 mg/L, pH value is 5.8 to be made.
Above-mentioned MS minimal medium includes a great number of elements (potassium nitrate 1900, ammonium nitrate 1650, potassium dihydrogen phosphate 170, seven water
Magnesium sulfate 370, calcium chloride dihydrate 440, unit mg/L), microelement (potassium iodide 0.83, boric acid 6.2, four water manganese sulfates
22.3, white vitriol 8.6, Sodium Molybdate Dihydrate 0.25, cupric sulfate pentahydrate 0.025, CoCL2 6H2O 0.025, unit mg/
L), molysite (disodium ethylene diamine tetraacetate 37.25, ferrous sulfate heptahydrate 27.85, unit mg/L), inositol (100mg/L), dimension
Raw element (thiamine hydrochloride 0.1, puridoxine hydrochloride 0.5, niacin 0.5, unit mg/L), glycine (2mg/L), sucrose (30g/
L), agar (7g/L), specific formula reference: Murashige T, Skoog F. A revised medium for rapid
growth and bioassays with tobacco tissues cultures. Physiol Planta, 1962, 15:
473-479。
The above-mentioned phonetic peptide in viral inhibitors Ningnan (or the phonetic peptide in Ningnan) buys manufacturer are as follows: Shanghai federation chemical industry has
Responsible company is limited, its total active constituent content in the phonetic peptide in product Ningnan: 68%, including Ningnanmycin content: 8%, phonetic peptide is mould
Cellulose content 30%, viral A content: 30%, dosage form: aqua.
, investigation and data statistics: inoculation 60 days after count, count planting percent.
As a result with analysis
2.1, different virus inhibitor handles the influence to shoot apical meristem planting percent
After 4 kinds of viral inhibitors processing (lentinan, the phonetic peptide in Ningnan, toxic fluoride phosphate, amino-oligosaccharide) processing, as a result table
Bright: after different inhibitor processing, the stem-apex Meristem culture planting percent of Xu-shu No.22 is declined slightly compared with control, but Ningnan is phonetic
After peptide processing, planting percent still can achieve 38.2%, the highest in the processing of all viral inhibitors.And the stem apex of abundant Xu's potato 20
Sediment diversion ratio planting percent, after with the phonetic peptide processing of amino-oligosaccharide and Ningnan, planting percent relatively control is significantly improved,
Especially planting percent is improved compared with control close to 9%(such as Fig. 1) up to 47.3% after the phonetic peptide processing in Ningnan.
, twice stem-apex Meristem culture seedling SPFMV and SPCSV detection
After the life of SPFMV and SPCSV association infects sweet potato altogether, discovery SPFMV virus relative difficult removing.As shown in Fig. 2, SPCSV is logical
Crossing a stem-apex Meristem culture removal efficiency can reach 100%, but the removal efficiency of SPFMV is only capable of reaching 60%.It is more through mushroom
After sugar, the processing of the phonetic peptide in Ningnan, toxic fluoride phosphate, amino-oligosaccharide,
SPFMV removal efficiency after different virus inhibitor infects two kinds of virus association lifes altogether has different degrees of raising, abundant Xu
After the lentinan of potato 20 and the phonetic peptide processing of Ningnan, SPFMV removal efficiency improves about 20% compared with control；Xu-shu No.22 Ningnan is phonetic
After peptide and amino-oligosaccharide processing, viral removal efficiency also improves 25-30%(such as Fig. 3 compared with control).
In conjunction with stem-apex Meristem culture planting percent and viral removal effect, 800 times of dilutions of the phonetic peptide in Ningnan are combined twice
Stem-apex Meristem culture can effectively remove SPFMV and SPCSV.
Claims (1)
1. a kind of sweet potato health seedling cultural method for removing sweet potato viruses complex SPVD, it is characterised in that: choose infection
SPVD virus plant, then by stem-apex Meristem culture, utilizes again through the phonetic peptide immersion treatment in viral inhibitors Ningnan
Regenerated plant carries out stem-apex Meristem culture, cultivates the sweet potato health seedling that removing sweet potato viruses complex SPVD infects；
Concrete operations sequentially include the following steps:
(1) configuration of the phonetic 800 times of dilutions of peptide in Ningnan: taking the total active constituent content of 2.5 ml is 68% viral inhibitors Ningnan
Phonetic peptide solution adds Hongland nutrient solution to be settled to 2 L spare；
(2) clip sweet potato viruses complex SPVD infects 25 cm of sweet potato sprout band stem apex stem section, is incubated at the phonetic peptide in above-mentioned Ningnan
In 800 times of dilutions, 96 h of incubated at room temperature；
(3) stem-apex Meristem culture: the 3 cm young tender stem tips in Ningnan 800 times of dilution cultures of phonetic peptide are taken, are rushed through tap water
It washes 5-10 minutes, Tween-80 is added to clean 2-3 min, after then rinsing 2-3 min with tap water again, then with 0.1% hypochlorous acid
Sodium concussion 20 min of sterilizing, then rinsed 3-4 times with sterile purified water, it is mitogenetic that 1-2 phyllopodium stem apex of band is cut under anatomical lens
Tissue is incubated at induction seedling culture medium and obtains first time stem-apex Meristem culture regeneration plant after cultivating seedling, chooses
The regeneration plant carries out removing stem-apex Meristem culture again, and culture medium is still above-mentioned induction seedling culture medium, obtains second
Secondary stem-apex Meristem culture regeneration plant；
(4) sweet potato viruses complex SPVD is detected: the sweet potato health for choosing second of stem-apex Meristem culture regeneration plant is planted
Strain, carries out SPVD to it using RT-PCR and ELISA and infects detection；
The induction seedling culture medium refers to adds 6-BA, 1 mg/L in MS minimal medium, NAA, 0.01 mg/L, TDZ,
0.5 mg/L, pH value 5.8.
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| CN113249403A (en) * | 2021-03-31 | 2021-08-13 | 江苏师范大学 | Breeding method of sweet potatoes with SPVD resistance |
| CN113317320A (en) * | 2021-04-26 | 2021-08-31 | 山东省农业科学院作物研究所 | Biological agent for improving virus-free rate of sweet potatoes and application thereof |
| CN114431143A (en) * | 2022-02-09 | 2022-05-06 | 廊坊市农林科学院 | Improved MS culture medium and preparation method and application thereof |
| CN114982635A (en) * | 2022-06-17 | 2022-09-02 | 紫云自治县紫香源农林科技有限责任公司 | Detoxified miniature sweet potato propagation method |
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| CN114431143A (en) * | 2022-02-09 | 2022-05-06 | 廊坊市农林科学院 | Improved MS culture medium and preparation method and application thereof |
| CN114982635A (en) * | 2022-06-17 | 2022-09-02 | 紫云自治县紫香源农林科技有限责任公司 | Detoxified miniature sweet potato propagation method |
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