CN109380216A - A kind of cryopreservation and renewal cultivation method of oryza officinalis stem apex - Google Patents
A kind of cryopreservation and renewal cultivation method of oryza officinalis stem apex Download PDFInfo
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- CN109380216A CN109380216A CN201811280227.0A CN201811280227A CN109380216A CN 109380216 A CN109380216 A CN 109380216A CN 201811280227 A CN201811280227 A CN 201811280227A CN 109380216 A CN109380216 A CN 109380216A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N3/00—Preservation of plants or parts thereof, e.g. inhibiting evaporation, improvement of the appearance of leaves or protection against physical influences such as UV radiation using chemical compositions; Grafting wax
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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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Abstract
The present invention relates to a kind of oryza officinalis band stem apex young shoot cryopreservation and regeneration plant methods to be specifically related to a kind of cryopreservation method of the oryza officinalis with stem apex young shoot.The method of platymiscium cell engineering field, cryopreservation and regeneration plant includes the following steps: the sampling of 1. fields and tissue culture Establishing；2. the detection of endophyte；3. the induction of Multiple Buds；4. the preculture of Multiple Buds；5. the embedding of Multiple Buds；6. the loading of Multiple Buds and vitrified processing；7. freezing and washing of thawing；8. renewal cultivation.Present invention firstly provides cryopreservation is successfully carried out to oryza officinalis band stem apex young shoot, wild rice restoration ecosystem is in order after preservation.There is good reference value to the cryopreservation of wild rice and gramineae plant.
Description
Technical field
The present invention relates to a kind of store methods of oryza officinalis stem apex, specifically, are related to a kind of oryza officinalis band
The cryopreservation method of stem apex young shoot.Platymiscium cell engineering field.
Background technique
Oryza officinalis is one of 3 kinds of wild rices originating in China, since long term growth is under severe natural conditions, medicine
It is subjected to the natural selection of various natural calamities and poor environment with wild rice, is a kind of very rare and important Wild rice germplasm
Resource is subjected to the natural selection of various disasters and environment, forms variation type abundant due to being chronically at wild state,
There is stronger resistance to rice pest, has stronger adaptability to environment, and there is high-quality, high yield, nitrogen phosphorus efficiency to utilize, is wide
The excellent genes such as affine and male sterility are the important genetic resources of rice breeding and improvement, have high scientific research valence
Value.The economic activity of current aggravating circumstances and unfavorable weather conditions and mankind's modern times explosion rapidly results in wild rice
Habitat loss and deterioration, habitat are sharply reduced；Furthermore wild rice has that stigma appearing, outcrossing seed-setting rate be higher, seed is stopped again
The biological characteristics that moultinism is strong, seed holding is strong, germination percentage is low.These factors make oryza officinalis kind face the danger of extinction, right
The protection of wild rice genetic diversity is very urgent.Currently, mainly having in-situ protection to the safeguard measure of oryza officinalis and moving
Ground protection (i.e. with the germplasm village, often used in village names of Seed storage, the field gene bank saved with seedling stem).
The culture of plant tissue and cell is that the preserving seed of plant opens new approach.Tissue culture can be big rapidly
Amount breeding, regeneration plant can keep original hereditary capacity.And under cryopreservation (- 196 DEG C), the biochemistry of plant is living
Property almost stop, the physiology in storage and heredity variation can control in bottom line, avoid field save vulnerable to
Effect of Natural Disaster and cause blastation or destruction and tissue cultures save in because multiple subculture causes inhereditary feature to make a variation
Or the danger of pollution, it is considered to be the optimal selection of plant genetic resources long-term preservation.
About the research of plant cryopreservation, constantly make progress in terms of the diversification and summary of Techniques of preserving, energy
The plant species number enough saved also increases constantly, but majority concentrates on the woody plant such as apple, cherry, citrus, banana, cassava
The cryopreservation of plant genetic resources based on the economic plants such as object and potato, sweet potato, strawberry.The ultralow temperature of rice is protected
It deposits after reporting the cryopreservation of rice suspension cell first from Sala in 1979 etc., correlative study focuses mostly in tissue culture
On suspension cell line, callus, protoplast and embryoid, the report frozen about rice complete organ (such as stem apex) is only
See Zhang Zhihong etc. 2000 and ultralow temperature is carried out to the adventitious bud that rice monoploid children's fringe in vitro culture induces using programmed cooling method
It saves, obtains regeneration plant.The cryopreservation technology relevant report of wild rice is less and concentrates on the country, such as Yin Xiaohui
It is obtained for the first time in common wild-rice, wide leaf wild rice and the research of tumor grain Callus From Wild Rice cryopreservation within 1996
Regeneration plant after freezing, the experiment proves that the cryopreservation of wild rice children's fringe isolated culture is having for Oryza resource conservation
Effect approach.Tan Guangxuan etc. induces plant, directly progress cryopreservation to wild rice children's fringe in vitro culture using programmed cooling method,
The danger that callus phase reduces hereditary variation is avoided, but albefaction is dead after a week for the green bud generated.
Wild rice complete organ's freezes, and will be the emphasis studied from now on, just due to wild rice shoot apical meristem volume
It is small, physiological status is fragile, be difficult to survive in preservation.Woods field etc. is adopted a series of measures for the first time, to improve the resistance of stem apex, is reduced
Damage in each step, and delivered directly and the freezing step of non-dependent programmed cooling instrument using liquid nitrogen, establish common wild-rice
Stem apex cryopreservation system, and (patent No.: ZL 201610131944.1) patented in 2018, but still have scarce
Point: 1. since wild rice explant is Field sampling, and endophytic bacterial contamination is difficult to control.2. due to by young tender stem tip by close
It is separated in the leaf of package, stem apex strips step to operator's technical requirements height, great work intensity, and holds during stripping
Mechanical wounding easily is caused to stem apex, the scale for being unfavorable for germ plasm resource saves.3. from Field sampling to being suitable for the material frozen
It is too long to obtain the period, it is difficult to which the material for obtaining a large amount of neat and consistents is unfavorable for the quantity frozen and quality control.
Summary of the invention
The object of the present invention is to provide a kind of cryopreservation methods of oryza officinalis stem apex, while also providing a kind of medicine
With the renewal cultivation method after the cryopreservation of wild rice stem apex.
In order to achieve the object of the present invention, a kind of oryza officinalis band stem apex young shoot cryopreservation method of the present invention, packet
Include following steps:
The sampling of the field step 1 and tissue culture Establishing: taking rhizomes, carefully washes away root attachment silt, is soaked with 1% carbendazim
Bubble is after 1 hour, and in clear water culture 10-20 days, the gas for taking aerial part newly to germinate was sprouted；Be inoculated with after routine disinfection, every 30 days after
Generation 1 time；
The detection of step 2 .. endophyte: tissue-cultured seedling is transferred to containing 10 g L-1Glucose and 5 g L-1The culture of yeast extract
It is cultivated 7-10 days in base, chooses untainted tissue-cultured seedling into being inoculated on the culture medium containing BA and NAA, every 30 days subcultures 1 time.
Step 3 inducing clumping bud: subculture 30 days sterile tissue-cultured seedling are taken, 2-3mm stem apex is stripped, is put into containing TDZ0.5-
5mgL-1MS culture medium in, induce within about 5-7 days the Multiple Buds of 2-3mm.
Step 4 Multiple Buds preculture: Multiple Buds band base portion is transferred to 0.3molL containing sucrose-1And ABA0.5-5mgL-1's
After cultivating 1-3 days on culture medium, it is transferred to 0.7molL-1And ABA0.5-5mgL-1Culture medium on preculture 1-3 days.
The embedding of step 5 Multiple Buds: the bud of 2-3mm is cut under anatomical lens, in 2-4% without calcium ion sodium alginate soln
In gently dip in, i.e., be neatly placed in the metal net shaped carrier strip of 5mm × 20mm, 5-10 bud in every carrier strip.By the loading with bud
Item, which immerses, contains 0.1-0.3% calcium chloride, 0.4 molL-1Sucrose and 2moL-1The MS fluid nutrient medium of glycerol (is free of NH4 +) in,
After 10-30min is fixed, carrier strip is sucked into surplus liquid on aseptic filter paper.
Step 6 Multiple Buds load and vitrifying processing: the carrier strip with bud fixed being put in preprocessing solution and (is contained
There is 0.4 molL-1And glycerol 2moL-1MS fluid nutrient medium, be free of NH4 +) in load 16hr after be transferred to vitrifying protective agent
PVS2(is free of NH4 +) in carry out 2-5hr ice bath processing,
Step 7 freezing and washing of thawing: the carrier strip with bud is put into liquid nitrogen rapidly and is saved.
The present invention also provides the renewal cultivation methods after a kind of cryopreservation of oryza officinalis stem apex, and step is such as
Under:
Step a. is removed from liquid nitrogen the carrier strip with bud when thawing, and is immediately placed in 1.2molL-1(be free of NH4 +) 1/2MS liquid
Middle quick-thawing 2min in body culture medium, and 20min is washed in the medium.
Step b. renewal cultivation: 1/2MS culture medium hormone-free is placed on (no for blotting on the carrier strip filter paper with bud
Containing NH4 +) in dark culture 1 day, be transferred to the culture medium containing BA and NAA (without NH4 +) on dark culture.It is transferred to after stem apex regeneration
Culture medium containing BA and NAA (contains NH4 +) illumination cultivation.
The present invention successfully carries out cryopreservation to oryza officinalis band stem apex young shoot for the first time, and wild rice restores life after preservation
Length is in order.
It since wild rice explant is Field sampling, has the disadvantage that, (1) endophytic bacterial contamination is difficult to control, and (2) are wild
The stem-tip tissue of raw rice has the characteristics that tiny, tender, and by outer protective leaf tight layer by layer, strips in stem apex, preculture
And frangibility and come to harm in glass frozen preservation operation, it is also not easy to regenerate or generate Albino Seedling in renewal cultivation behind.
(3) from Field sampling to being suitable for that obtain the period too long for the material frozen, it is difficult to which the material for obtaining a large amount of neat and consistents is unfavorable for freezing
Quantity and the quality control deposited.According to above-mentioned technological deficiency, the measure that the present invention takes has: (1) is taken off at bacterium by early period
Reason, induction gas is sprouted and the series of steps of endophyte detection, further controls endophytic bacterial contamination；(2) is lured using de- bacterium stem apex
Grow thickly young shoot of the derived oryza officinalis containing stem apex, obtains the material of a large amount of neat and consistents；(3) will grow thickly young shoot as jelly
Material is deposited, the cumbersome technological means that wild rice stem apex strips is eliminated, finally obtains the regeneration plant of oryza officinalis.To foundation
The wild rice cryopreservation system of convenient stability and high efficiency is conducive to the preservation of Exploration of Wild Rice Germplasm Resources scale, to construct wild resource
Library has good reference value.
Grow thickly young shoot of the oryza officinalis containing stem apex that the present invention is gone out using de- bacterium Stem tip induction is saved as frozen material
It further reduced operation difficulty, simplify operating process and processing time, and by taking off bacterium processing early period, induction gas is sprouted
And the series of steps of endophyte detection, endophytic bacterial contamination is further controlled, convenient and efficient is conducive to scale preservation, to build
Vertical wild rice cryo-conservation resources bank has made technological reserve.
Detailed description of the invention
Fig. 1 oryza officinalis cryopreservation flow chart；
Fig. 2 wild rice inducing clumping bud and Cryopreservation；
In Fig. 2: a: the raw adventitious bud of the gas of wild rice；B: oryza officinalis Multiple Buds；C: the carrier strip of fixed wild rice Multiple Buds；
D: renewal cultivation regeneration bud after jelly.
Specific embodiment
Following embodiment is merely to illustrate the present invention, but is not intended to limit the scope of the invention.
Embodiment 1, the acquisition of wild rice aseptic seedling and de- bacterial examination are surveyed
It takes the subterranean stem band of wild rice to germinate the stem section of bud, carefully washes away root attachment silt, impregnated 1 hour with 1% carbendazim
Afterwards, in clear water culture 10-20 days, the gas for taking aerial part newly to germinate is sprouted, under tap water flowing water after repeated flushing about 1hr,
With 70% alcohol disinfecting, 1 min, 0.1% mercuric chloride sterilizes 15 min, aseptic water washing several times after, about 2 are stripped under anatomical lens
The stem apex of mm is inoculated in containing 4 mgL-1BA and 0.5 mgL-1Multiple Buds are induced on the MS culture medium of NAA.Cultivation temperature
It is 25 ± 2 DEG C, 12 hd of illumination-1, 36 μm of ol m of intensity of illumination-2 s-1.After inducing Multiple Buds, it is transferred to containing 10 g L-1
Glucose and 5 g L-1It is cultivated 7-10 days in the culture medium of yeast extract, chooses untainted Multiple Buds and cut off and be seeded in and contain
2mg·L-1BA and 0.5 mgL-1Squamous subculture on the MS culture medium of NAA, every 30 days subcultures 1 time.
The Multiple Buds acquisition of embodiment 2, wild rice
The resulting tissue-cultured seedling of Example 1, takes subculture 30 days sterile tissue-cultured seedling, strips 2-3mm stem apex, be put into containing TDZ4mgL-1MS culture medium in, induce within about 5-7 days the Multiple Buds of 2-3mm.
Embodiment 3, wild rice Multiple Buds device fix
With tweezers 5mm will be neatly placed in 2-4% without gently dipping in calcium ion sodium alginate soln by the Multiple Buds of preculture
In the metal net shaped carrier strip of × 20mm, 10 buds in every carrier strip.By with stem apex carrier strip immerse containing 0.2% calcium chloride,
0.4 molL-1Sucrose and 2moL-1The MS fluid nutrient medium of glycerol (is free of NH4 +) in, after 20min is fixed, by carrier strip in nothing
Surplus liquid is sucked on bacterium filter paper.
Example IV is the process step and growth conditions embodiment of stem apex cryopreservation and renewal cultivation
The step of Fig. 1 is oryza officinalis cryopreservation flow chart, shown referring to Fig.1, is saved is as follows:
Rhizomes is taken, root attachment silt is carefully washed away and, in clear water culture 10-20 days, is taken after being impregnated 1 hour with 1% carbendazim
The gas that aerial part newly germinates is sprouted；It is inoculated with after routine disinfection, every 30 days subcultures 1 time；Tissue-cultured seedling is transferred to containing 10 g L-1
It is cultivated 7-10 days in the culture medium of glucose and 5 g L-1 yeast extracts, chooses untainted tissue-cultured seedling into being inoculated into containing BA
And on the culture medium of NAA, every 30 days subcultures 1 time.The sterile tissue-cultured seedling for taking subculture 30 days, strips 2-3mm stem apex, is put into and contains
In the MS culture medium of TDZ0.5-5mgL-1, the Multiple Buds of 2-3mm are induced within about 5-7 days.Multiple Buds band base portion is transferred to containing sugarcane
After cultivating 1-3 days on the culture medium of sugared 0.3molL-1 and ABA0.5-5mgL-1, it is transferred to 0.7molL-1 and ABA0.5-5mgL-1
Culture medium on preculture 1-3 days.The bud that 2-3mm is cut under anatomical lens, in 2-4% without light in calcium ion sodium alginate soln
It dips in, i.e., is neatly placed in the metal net shaped carrier strip of 5mm × 20mm, 5-10 bud in every carrier strip.By the carrier strip leaching with bud
Enter the MS fluid nutrient medium containing 0.1-0.3% calcium chloride, 0.4 molL-1 sucrose and 2moL-1 glycerol (without NH4+) in, warp
After 10-30min is fixed, carrier strip is sucked into surplus liquid on aseptic filter paper.The carrier strip with bud fixed is put in pre-
(the MS fluid nutrient medium containing 0.4 molL-1 and glycerol 2moL-1 is free of NH4 to processing solution+) in load 16hr after be transferred to glass
Glass protective agent PVS2(is free of NH4+) in carry out 2-5hr ice bath processing, the carrier strip with bud is put into liquid nitrogen rapidly and is protected
It deposits.The carrier strip with bud is removed from liquid nitrogen when defrosting, be immediately placed in 1.2molL-1 (is free of NH4+) 1/2MS Liquid Culture
Middle quick-thawing 2min in base, and 20min is washed in the medium.It will blot and be placed on without sharp on carrier strip filter paper with bud
The 1/2MS culture medium of element (is free of NH4+) in dark culture 1 day, be transferred to the culture medium containing BA and NAA (without NH4+) on secretly train
It supports.The culture medium containing BA and NAA is transferred to after stem apex regeneration (containing NH4+) illumination cultivation.).
Fig. 2 is the state diagram of wild rice inducing clumping bud and Cryopreservation, as shown in Fig. 2, the steps include:
A: the raw adventitious bud of the gas of wild rice: taking the subterranean stem band of wild rice to germinate the stem section of bud, carefully washes away root attachment mud
Sand, after being impregnated 1 hour with 1% carbendazim, in clear water culture 10-20 days, the gas that aerial part newly germinates was sprouted.
B: oryza officinalis Multiple Buds: gas sprout it is sterilized after the sterile tissue-cultured seedling of induction, take subculture 30 days sterile tissue cultures
Seedling strips 2-3mm stem apex, is put into the MS culture medium containing TDZ4mgL-1, induces within about 5-7 days the Multiple Buds of 2-3mm.
C: the carrier strip of fixed wild rice Multiple Buds: the Multiple Buds of 2-3mm are in 2-4% without calcium ion sodium alginate soln
In gently dip in, be neatly placed in the metal net shaped carrier strip of 5mm × 20mm, 10 buds in every carrier strip.By the carrier strip with stem apex
It immerses and contains 0.2% calcium chloride, 0.4 molL-1Sucrose and 2moL-1The MS fluid nutrient medium of glycerol (is free of NH4 +) in, through 20min
After fixation, carrier strip is sucked into surplus liquid on aseptic filter paper.
D: it renewal cultivation regeneration bud after jelly: after defrosting and washing, is placed on being blotted on the carrier strip filter paper with bud not
1/2MS culture medium containing hormone (is free of NH4 +) in dark culture 1 day, be transferred to the culture medium containing BA and NAA (without NH4 +) on it is dark
Culture.The culture medium containing BA and NAA is transferred to after stem apex regeneration (containing NH4 +) illumination cultivation.
Claims (3)
1. a kind of cryopreservation method of oryza officinalis stem apex, includes the following steps:
The sampling of the field step 1 and tissue culture Establishing: taking rhizomes, washes away root attachment silt, impregnates 1 with 1% carbendazim
After hour, in clear water culture 10-20 days, the gas for taking aerial part newly to germinate was sprouted；It is inoculated with after routine disinfection, every 30 days subcultures 1
It is secondary;
The detection of step 2 endophyte: tissue-cultured seedling is transferred to containing 10 g L-1Glucose and 5 g L-1The culture medium of yeast extract
Middle culture 7-10 days, chooses untainted tissue-cultured seedling into being inoculated on the culture medium containing BA and NAA, and every 30 days subcultures 1 time;
Step 3 inducing clumping bud: the subculture 30 days sterile tissue-cultured seedling of sterile tissue-cultured seedling are taken, 2-3mm stem apex is stripped, is put into and contains
TDZ0.5-5mgL-1MS culture medium in, induce within about 5-7 days the Multiple Buds of 2-3mm；
Step 4 Multiple Buds preculture: Multiple Buds are transferred to the training of 0.3molL-1 containing sucrose and ABA0.5-5mgL-1 with base portion
It supports after being cultivated 1-3 days on base, is transferred on the culture medium of 0.7molL-1 and ABA0.5-5mgL-1 preculture 1-3 days;
The embedding of step 5 Multiple Buds: cutting the bud of 2-3mm under anatomical lens, in 2-4% without light in calcium ion sodium alginate soln
It dips in, is neatly placed in the metal net shaped carrier strip of 5mm × 20mm, 5-10 bud in every carrier strip immerses the carrier strip with bud
In MS fluid nutrient medium (being free of NH4+) containing 0.1-0.3% calcium chloride, 0.4 molL-1 sucrose and 2moL-1 glycerol, through 10-
After 30min is fixed, carrier strip is sucked into surplus liquid on aseptic filter paper；
Step 6 Multiple Buds load and vitrifying processing: the carrier strip with bud fixed being put in preprocessing solution and is loaded
Vitrifying protective agent PVS2(is transferred to after 16hr without NH4+) in carry out 2-5hr ice bath processing；
Step 7 freezing and washing of thawing: the carrier strip with bud is put into liquid nitrogen rapidly and is saved.
2. cryopreservation method of the oryza officinalis according to claim 1 with stem apex young shoot, which is characterized in that institute
It states in preprocessing solution as the MS fluid nutrient medium containing 0.4 molL-1 and glycerol 2moL-1, is free of NH4+。
3. a kind of renewal cultivation method after oryza officinalis stem apex cryopreservation comprising following steps:
Step a. is removed from liquid nitrogen the carrier strip with bud when thawing, and be immediately placed in 1.2molL-1 (is free of NH4+) 1/2MS liquid
Middle quick-thawing 2min in body culture medium, and 20min is washed in the medium;
Step b. renewal cultivation: it will be blotted on the carrier strip filter paper with bud and be placed on 1/2MS culture medium hormone-free and (be free of
NH4+) in dark culture 1 day, be transferred to the culture medium containing BA and NAA (without NH4+) on dark culture, be transferred to and contain after stem apex regeneration
The culture medium of BA and NAA (contains NH4+) illumination cultivation.
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| CN110800734A (en) * | 2019-11-21 | 2020-02-18 | 上海市农业生物基因中心 | Ultra-low temperature preservation method for wild rice stem tips |
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| 章志宏: "水稻单倍体不定芽超低温保存和植株再生及其遗传稳定性研究", 《武汉植物学研究》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110800734A (en) * | 2019-11-21 | 2020-02-18 | 上海市农业生物基因中心 | Ultra-low temperature preservation method for wild rice stem tips |
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