CN110122335A - The abductive approach of Xu Xiang Kiwifruit Tissue Culture seedling salt-tolerant mutant - Google Patents
The abductive approach of Xu Xiang Kiwifruit Tissue Culture seedling salt-tolerant mutant Download PDFInfo
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- CN110122335A CN110122335A CN201910566729.8A CN201910566729A CN110122335A CN 110122335 A CN110122335 A CN 110122335A CN 201910566729 A CN201910566729 A CN 201910566729A CN 110122335 A CN110122335 A CN 110122335A
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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
- A01H4/001—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
- A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
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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 belongs to agricultural breeding technical fields, the specially abductive approach of Xu Xiang Kiwifruit Tissue Culture seedling salt-tolerant mutant, this method is using the fresh and healthy blade of ' Xu Xiang ' Kiwi berry as explant, establish sterile tissue culture system, EMS mutagenesis plant is obtained using the method that tissue culture technique and EMS mutagenesis combine, using NaCl as Salt Tolerance agent, salt-tolerant mutant screening is carried out to EMS mutagenesis seedling.Salt tolerant ' Xu Xiang ' kiwi fruit plant that this method obtains is suitable for salt-soda soil environment, to improve land utilization ratio and and economic benefit, and have great importance to agriculture and forestry productivity and its sustainability.
Description
Technical field
The invention belongs to agricultural breeding technical fields, specially the induction side of Xu Xiang Kiwifruit Tissue Culture seedling salt-tolerant mutant
Method.
Background technique
' Xu Xiang ' Kiwi berry is the perennial defoliation liana of Actinidiaceae Actinidia, is comprehensive product in Kiwifruit
Best one of the kind of matter.' Xu Xiang ' Kiwi berry is mainly planted in Jiangsu Province, and plantation is also introduced a fine variety in the various regions such as Shaanxi, Henan,
Its salt tolerance is to be improved.
China is one of the countries with the most serious ... in being influenced by saline Land, can be resistance to by cultivating to the prevention and treatment of saline-alkali soil
Salt kind is realized；Salt-resistant plant breeding is had become with the technology of the technology screening plant soma salt-tolerant mutant of tissue cultures
The important topic of area research, but still belong to empty about the research of ' Xu Xiang ' Kiwifruit Tissue Culture seedling salt-tolerant mutant induction scheme at present
It is white.
Summary of the invention
For the bad problem of existing ' Xu Xiang ' Kiwifruit Cultivars salt tolerance, the present invention proposes a kind of Xu Xiang Kiwi berry group
Train the abductive approach of seedling salt-tolerant mutant.
The abductive approach of Xu Xiang Kiwifruit Tissue Culture seedling salt-tolerant mutant of the invention, it is characterised in that pass through following steps reality
It is existing:
1) sterile system is established:
Picking health and eugonic Kiwi berry blade, cleaning blade surface, while to superclean bench, culture medium and other
Experimental article carries out disinfection, and the thimerosal combination uses 75% alcohol treatment 15 seconds, and mercuric chloride is handled 4 minutes；With sterile after disinfection
Water adequately rinses four to five times, and each sterile water consumption is to flood explant；
To ' Xu Xiang ' Kiwi berry explant Disinfection Effect, there were significant differences for difference disinfection treatment.For the death rate, at 75% ethyl alcohol
Reason 15 seconds, it is 0 minimum that mercuric chloride, which handles 4 minutes death rates, but the combination of optimal thimerosal is considered as its survival rate and pollution rate, i.e.,
Thimerosal combines 75% alcohol treatment 15 seconds, and mercuric chloride is handled 4 minutes, and for survival rate mean value up to 95.00%, pollution rate mean value is 0, dead
Rate mean value is 5.00%.
2) callus induction:
Blade will be cut into the square of 1cm × 1cm, blade face is inoculated into culture medium upward, and the culture medium is MS+
TDZ1.0mg/L+0.15IBAmg/L；Pressing lightly on makes it be bonded culture medium, is placed in culturing room and cultivates after inoculation, first carries out
5 days shading dark treatments, then illumination cultivation 25 days, form callus；
The induction of ' Xu Xiang ' Cultured Actinidia deliciosa Calli is closely related with the basic element of cell division and auxin, needs to balance between the two
It adjusts；The culture medium of this patent is used through testing, callus color is green, it is very fast in the lumps and growth of densification, more
It is preferable to hurt quality, callus is induced up to 100%.
3) EMS is induced:
Using mixed training method, the callus of survival is inoculated into culture medium, the culture medium is agar 4.5g/L+ sucrose
30.0g/L, pH=5.8-6.0；The EMS filter sterilizing solution that phosphate buffer is configured, the EMS solution concentration are 0.4g/
L is added culture medium using additive process, grows 20 days, obtains the plant induced through EMS；
EMS concentration is one of key problem of the present invention: temperature 50 C or so also not solidified proliferation is added in the EMS of above-mentioned concentration
It is shaken in culture medium uniformly, accesses the good callus of growth potential again after culture medium solidification, callus is average after 20 days
Lethality is 53.33%, and the callus under surviving also almost has half browning death, therefore culture medium is added using additive process,
Up to semilethal after growth 20 days.
4) adventitious bud inducing breaks up:
Well-grown Callus material is inoculated into culture medium, the culture medium is MS+6-BA1.0mg/L+
NAA0.2mg/L is placed in culturing room after inoculation and cultivates；
' Xu Xiang ' Kiwi berry adventitious bud inducing differentiated result need to consider sprout number, value-added coefficient, average height of seedling and the number that averagely sprouts,
Comprehensive height of seedling and hormone dosage situation, above-mentioned processing combination Corticosteroids amount is less, and the adventitious bud number that averagely sprout is 6.43, is averaged
The high 1.37cm of bud.
5) salt-tolerant plants screen:
By after the callus under EMS inducing survival is trained intact plant, access in the salt culture medium of salinity 1.0%, the training
Supporting base is MS culture medium+1mg/LTDZ+0.15mg/LIBA+ sucrose 25g/L+ agar 4.5g/L, pH 5.8, is containing salt culture medium
After growth 15 days, then it is transferred in salt-free proliferated culture medium and grows 15 days, so progress Salt Tolerance；3 week of alternate culture
The seedling survived is gone to salt-free proliferated culture medium rejuvenation, obtains fragrant macaque peach seedling salt-tolerant plants by the phase.
After accessing salt culture medium 20 days of above-mentioned salinity, the lethality of ' Xu Xiang ' kiwi fruit plant is up to 95%, this concentration
Critical concentration, the mutant salt tolerance under screening survival are stronger；
Whole nation saline alkali land area is larger at present, has wide development space to the development and utilization in salt-soda soil.' Xu Xiang ' Kiwi berry is comprehensive
It is good to close character, economic value is high, but its salt tolerance has room for promotion.It is induced in tissue culture system with EMS, use is made with NaCl
For salt tolerant selective agent, and then salt-tolerant mutant screening is screened, cultivates ' Xu Xiang ' macaque peach seedling of higher salt resistance ability.Through testing
Verifying, the mutant plant and normal plant that obtain through abductive approach of the invention are placed in the salt content of 95% lethasl concentration together
In 1.0% culture medium, normal plant is by apparent salt damage after a week, and mutant plant normal growth, is tentatively obtained with this prominent
Becoming plant has salt tolerance.After the invention is applied, ' Xu Xiang ' Kiwi berry popularizing planting area will be improved, simultaneously also
Can effectively develop and use salt-soda soil, improve land utilization ratio and and economic benefit, to agriculture and forestry productivity and its sustainability
Have great importance.
6) rooting induction:
The good tissue culture plant inoculation of growth potential is chosen into culture medium, the culture medium is MS+IBA0.75mg/L+AC0.30g/
L, when inoculation, remove callus, intercept stem section part, are placed in culturing room and cultivate after inoculation；
' Xu Xiang ' Kiwifruit Tissue Culture seedling rooting is based on auxin, and selection index system is strong, long action time, grows induction root more
Auxin IBA carries out rooting induction in combination with active carbon, and using above-mentioned culture medium, number of averagely taking root is 8.91, average root long
Up to 9.38cm, average rooting rate is up to 98.33%.
7) acclimatization and transplants:
Select fertile soil: garden mould: turfy soil presses the matrix of 1:1:1 mixing, and the vigorous normal tissue-cultured seedling of taking root of growing way is moved to certainly
Bottle cap is loosened after carrying out growth 10 days under right light, tissue culture bottle cap is opened after 5 days and is covered with gauze, after 5 days
It takes out tissue-cultured seedling flowing water to rinse the culture medium being attached on root well, and is carried out disinfection foam washing 5 minutes with carbendazim solution,
Surface moisture is dried under cool environment later, is planted to the different soil for carrying out antivirus processing with Bavistin in advance
In matrix, the soil matrix is selected containing several in fertile soil, garden mould, turfy soil or vermiculite, is placed in shady place, is used plastic lid
Flowerpot is covered, maintains the temperature at 26 ± 3 DEG C, sprays water mist humid control 80% or more；
Above-mentioned substrate combination average survival is combined up to 83.33% for optimum substrate；Fertile soil, garden mould, turf can be selected in matrix
One or more of in soil or vermiculite: vermiculite is loose porous, and quality is light, can absorb a large amount of water, is retained, holds the energy of fertilizer, heat absorption, heat preservation
Power is also relatively strong；Turfy soil is that plant debris rots to be formed, and it is strong that good water-retaining property stores fertile ability；Fertile soil rots to be formed by falling leaves, and contains
A large amount of mineral nutritions and organic substance；Garden mould stickiness is good, has certain fertility.Through testing, fertile soil and turfy soil are mixed and are used
When be more suitable for ' Xu Xiang ' Kiwi berry tissue-cultured seedling survival.The acclimatization and transplants effect of four kinds of matrix is ranked up: turfy soil > humic
Soil > garden mould > vermiculite.
Detailed description of the invention
Fig. 1 is upgrowth situation of ' Xu Xiang ' the Kiwi berry mutant plant on the culture medium of saliferous 1%.
Fig. 2 is that ' Xu Xiang ' Kiwi berry is normally placed in containing the growing state in 1.0% salt culture medium with mutant plant.
Specific embodiment
Embodiment 1: picking health and eugonic Kiwi berry blade, cleaning blade surface, at the same to superclean bench,
Culture medium and other experimental articles carry out disinfection, and the thimerosal combination uses 75% alcohol treatment 15 seconds, and mercuric chloride is handled 4 minutes；
It is adequately rinsed four to five times after disinfection with sterile water, each sterile water consumption is to flood explant；
Blade will be cut into the square of 1cm × 1cm, blade face is inoculated into culture medium upward, and the culture medium is MS+
TDZ1.0mg/L+0.15IBAmg/L；Pressing lightly on makes it be bonded culture medium, is placed in culturing room and cultivates after inoculation, first carries out
5 days shading dark treatments, then illumination cultivation 25 days, form callus；
Well-grown Callus material is inoculated into culture medium, the culture medium is MS+6-BA1.0mg/L+
NAA0.2mg/L is placed in culturing room after inoculation and cultivates；
The callus of survival is inoculated into culture medium, the culture medium is agar 4.5g/L+ sucrose 30.0g/L, pH=5.8-
6.0；The EMS filter sterilizing solution that phosphate buffer is configured, the EMS solution concentration are 0.4g/L, are added using additive process
Culture medium is grown 20 days, obtains the plant induced through EMS；
By after the callus under EMS inducing survival is trained intact plant, access in the salt culture medium of salinity 1.0%, the training
Supporting base is MS culture medium+1mg/LTDZ+0.15mg/LIBA+ sucrose 25g/L+ agar 4.5g/L, pH 5.8, is containing salt culture medium
After growth 15 days, then it is transferred in salt-free proliferated culture medium and grows 15 days, so progress Salt Tolerance；3 week of alternate culture
The seedling survived is gone to salt-free proliferated culture medium rejuvenation, obtains fragrant macaque peach seedling salt-tolerant plants by the phase；
The good tissue culture plant inoculation of growth potential is chosen into culture medium, the culture medium is MS+IBA0.75mg/L+AC0.30g/
L, when inoculation, remove callus, intercept stem section part, are placed in culturing room and cultivate after inoculation；
Select fertile soil: garden mould: turfy soil presses the matrix of 1:1:1 mixing, and the vigorous normal tissue-cultured seedling of taking root of growing way is moved to certainly
Bottle cap is loosened after carrying out growth 10 days under right light, tissue culture bottle cap is opened after 5 days and is covered with gauze, after 5 days
It takes out tissue-cultured seedling flowing water to rinse the culture medium being attached on root well, and is carried out disinfection foam washing 5 minutes with carbendazim solution,
Surface moisture is dried under cool environment later, is planted to the different soil for carrying out antivirus processing with Bavistin in advance
In matrix, the soil matrix is selected containing several in fertile soil, garden mould, turfy soil or vermiculite, is placed in shady place, is used plastic lid
Flowerpot is covered, maintains the temperature at 26 ± 3 DEG C, sprays water mist humid control 80% or more.
Claims (1)
1. the abductive approach of Xu Xiang Kiwifruit Tissue Culture seedling salt-tolerant mutant, it is characterised in that realized by following steps:
1) sterile system is established:
Picking health and eugonic Kiwi berry blade, cleaning blade surface, while to superclean bench, culture medium and other
Experimental article carries out disinfection, and the thimerosal combination uses 75% alcohol treatment 15 seconds, and mercuric chloride is handled 4 minutes；With sterile after disinfection
Water adequately rinses four to five times, and each sterile water consumption is to flood explant；
2) callus induction:
Blade will be cut into the square of 1cm × 1cm, blade face is inoculated into culture medium upward, and the culture medium is MS+
TDZ1.0mg/L+0.15IBAmg/L；Pressing lightly on makes it be bonded culture medium, is placed in culturing room and cultivates after inoculation, first carries out
5 days shading dark treatments, then illumination cultivation 25 days, form callus；
3) EMS is induced:
The callus of survival is inoculated into culture medium, the culture medium is agar 4.5g/L+ sucrose 30.0g/L, pH=5.8-
6.0；The EMS filter sterilizing solution that phosphate buffer is configured, the EMS solution concentration are 0.4g/L, are added using additive process
Culture medium is grown 20 days, obtains the plant induced through EMS；
4) adventitious bud inducing breaks up:
Well-grown Callus material is inoculated into culture medium, the culture medium is MS+6-BA1.0mg/L+
NAA0.2mg/L is placed in culturing room after inoculation and cultivates；
5) salt-tolerant plants screen:
By after the callus under EMS inducing survival is trained intact plant, access in the salt culture medium of salinity 1.0%, the training
Supporting base is MS culture medium+1mg/LTDZ+0.15mg/LIBA+ sucrose 25g/L+ agar 4.5g/L, pH 5.8, is containing salt culture medium
After growth 15 days, then it is transferred in salt-free proliferated culture medium and grows 15 days, so progress Salt Tolerance；3 week of alternate culture
The seedling survived is gone to salt-free proliferated culture medium rejuvenation, obtains fragrant macaque peach seedling salt-tolerant plants by the phase；
6) rooting induction:
The good tissue culture plant inoculation of growth potential is chosen into culture medium, the culture medium is MS+IBA0.75mg/L+AC0.30g/
L, when inoculation, remove callus, intercept stem section part, are placed in culturing room and cultivate after inoculation；
7) acclimatization and transplants:
Select fertile soil: garden mould: turfy soil presses the matrix of 1:1:1 mixing, and the vigorous normal tissue-cultured seedling of taking root of growing way is moved to certainly
Bottle cap is loosened after carrying out growth 10 days under right light, tissue culture bottle cap is opened after 5 days and is covered with gauze, after 5 days
It takes out tissue-cultured seedling flowing water to rinse the culture medium being attached on root well, and is carried out disinfection foam washing 5 minutes with carbendazim solution,
Surface moisture is dried under cool environment later, is planted to the different soil for carrying out antivirus processing with Bavistin in advance
In matrix, the soil matrix is selected containing several in fertile soil, garden mould, turfy soil or vermiculite, is placed in shady place, is used plastic lid
Flowerpot is covered, maintains the temperature at 26 ± 3 DEG C, sprays water mist humid control 80% or more.
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Cited By (2)
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| CN113728919A (en) * | 2021-09-03 | 2021-12-03 | 湖南省园艺研究所 | Method for obtaining homogeneous mutant of kiwi fruit callus |
| CN117074450A (en) * | 2023-08-18 | 2023-11-17 | 四川省自然资源科学研究院(四川省生产力促进中心) | Method for screening kiwi fruit mutant based on pollen morphology |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113728919A (en) * | 2021-09-03 | 2021-12-03 | 湖南省园艺研究所 | Method for obtaining homogeneous mutant of kiwi fruit callus |
| CN117074450A (en) * | 2023-08-18 | 2023-11-17 | 四川省自然资源科学研究院(四川省生产力促进中心) | Method for screening kiwi fruit mutant based on pollen morphology |
| CN117074450B (en) * | 2023-08-18 | 2024-04-16 | 四川省自然资源科学研究院(四川省生产力促进中心) | Method for screening kiwi fruit mutant based on pollen morphology |
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| CN110122335B (en) | 2022-03-22 |
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