CN105746351B - A kind of cryopreservation of wild rice stem apex and renewal cultivation method - Google Patents
A kind of cryopreservation of wild rice stem apex and renewal cultivation method Download PDFInfo
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- CN105746351B CN105746351B CN201610131944.1A CN201610131944A CN105746351B CN 105746351 B CN105746351 B CN 105746351B CN 201610131944 A CN201610131944 A CN 201610131944A CN 105746351 B CN105746351 B CN 105746351B
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Classifications
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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
-
- 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
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Environmental Sciences (AREA)
- Developmental Biology & Embryology (AREA)
- General Health & Medical Sciences (AREA)
- Health & Medical Sciences (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Plant Pathology (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Dentistry (AREA)
- Toxicology (AREA)
- Agronomy & Crop Science (AREA)
- Botany (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The cryopreservation and renewal cultivation method, step of a kind of wild rice stem apex have：The sterile tissue-cultured seedling of squamous subculture is taken, base portion is stayed to be put into without being cultivated in hormone MS culture mediums, base portion is cut off and newly sprouts plant, be transferred to containing 0.3molL‑10.5 5mgL of sucrose and abscisic acid‑1It is cultivated on culture medium, then is transferred to 0.7molL‑1With containing 0.5 5mgL of abscisic acid‑1Preculture on culture medium, strips stem apex, in 0.4 molL‑1Sucrose and glycerine 2moL‑1MS solid medium precultures, 2 4% without calcium ion sodium alginate soln in gently dip in, be placed in carrier strip, then immerse containing 0.1 0.3% calcium chloride, 0.4 molL‑1Sucrose and 2moL‑1In glycerine MS fluid nutrient mediums, after fixation, surplus liquid is sucked, is placed in preprocessing solution and handles, then be transferred to progress ice bath processing in vitrifying protective agent PVS2, then put into liquid nitrogen and preserve.Advantage is that store method is simple and easy to do, reliable and stable, and wild rice restoration ecosystem is in order after preservation.
Description
Technical field
The present invention relates to a kind of store methods of wild rice stem apex, specifically, are related to a kind of common wild-rice stem apex
Cryopreservation method.Meanwhile the invention further relates to the renewal cultivation method after the cryopreservation of wild rice stem apex, belong to
Field of plant cell engineering technology.
Background technology
Exploration of Wild Rice Germplasm Resources is the important composition part of Rice Resources, it saves rice-cultivating not or has disappeared
Gene, and with special merit and high disease and insect resistance characteristic, the excavation and preservation of beneficial gene, to improve
Yield and quality of rice has immeasurable value.Current rapid aggravating circumstances and unfavorable weather conditions are corroding
The genetic diversity of rice；The economic activity of mankind's modern times explosion results in wild rice habitat loss and deterioration, habitat
Drastically reduce；In addition wild rice has stigma appearing, the biological characteristics that outcrossing seed-setting rate is higher, seed shattering is strong again.This
A little factors make wild seed rice face the danger of extinction, very urgent to the protection of wild rice genetic diversity.At present, to wild
The safeguard measure of rice mainly has in-situ protection and in situ conservation, i.e., with the germplasm village, often used in village names of Seed storage, the field gene bank preserved with seedling stem.
The culture of plant tissue and cell opens new approach for the preserving seed of plant, and tissue culture can be big rapidly
Amount breeding, regeneration plant can keep original hereditary capacity.And cryopreservation（-196℃）Under, the biochemistry of plant is lived
Property almost stop, the physiology in storage and heredity variation can control in bottom line, avoid field preserve easily by
Effect of Natural Disaster and cause blastation or destruction and tissue cultures preserve in because multiple subculture causes inhereditary feature to make a variation
Or the danger of pollution, it is considered to be the optimal selection that plant genetic resources preserve for a long time.
About the research of plant cryopreservation, constantly make progress in terms of the diversification of Techniques of preserving and summary, energy
The plant species number enough preserved 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 from after Sala in 1979 etc. the first cryopreservation of report rice suspension cell, correlative study focuses mostly in tissue culture
On suspension cell line, callus, protoplast and embryoid, about rice complete organ（Such as stem apex）The report frozen is only
See that professor Zhang Zhihong waits to carry out the adventitious bud that rice monoploid children's fringe cultured in vitro induces using programmed cooling method in 2000
Cryopreservation obtains regeneration plant.The cryopreservation technology relevant report of wild rice is less and concentrates on the country, such as Yin Xiao
The head in common wild-rice, wide leaf wild rice and the research of knurl grain Callus From Wild Rice cryopreservation in 1996 such as brightness professor
It is secondary frozen after regeneration plant, the experiment proves that the cryopreservation of wild rice children's fringe isolated culture is Oryza resource conservation
Effective way.Professor Tan Guangxuan etc. induces plant using programmed cooling method to wild rice children's fringe cultured in vitro, is directly surpassed
Cord blood avoids the danger that callus phase reduces hereditary variation, 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, but due to wild rice shoot apical meristem volume
It is small, since the stem-tip tissue of wild rice has the characteristics that tiny, tender, physiological status is fragile, and close layer by layer by outer protective leaf
Package easily snaps off and comes to harm in stem apex strips, preculture and glass frozen preservation operate, is difficult to survive in preservation.Together
When, it is not easy to regenerate or generate Albino Seedling in renewal cultivation behind.Using the freezing method of program cooling to equipment requirement
It is higher.
Invention content
Wild rice shoot apical meristem is small, physiological status is fragile due to existing in the prior art, and is difficult into preservation
The phenomenon that living.The object of the present invention is to provide a kind of cryopreservation method of wild rice stem apex, while also provide a kind of wild
Defrosting renewal cultivation method after rice stem apex cryopreservation method.
In order to achieve the object of the present invention, the present invention provides a kind of cryopreservation method of wild rice stem apex, including
Following steps：
Subculture 3 months or more sterile tissue-cultured seedling is taken, top plant is cut off, stays base portion about 3cm, root about 1cm, which is put into, to be free of
The MS culture mediums of hormone.After culture 5-7 days, the new rudiment 0.5-1cm of base portion new will be sprouted after plant cuts off, will be transferred to base portion containing sugarcane
Sugared 0.3molL-1And abscisic acid 0.5-5mgL-1Culture medium on cultivate.After culture 7-10 days, it is transferred to 0.7molL-1And abscisic acid
0.5-5mgL-1Culture medium on preculture 3-7 days.Then the stem apex of 2-3mm is stripped under anatomical lens, is containing 0.4 molL-1And
Glycerine 2moL-1MS solid mediums on preculture 16 hours, wherein, contain 0.4 molL-1And glycerine 2moL-1MS solids
Without NH4+ in culture medium, the stem apex after preculture is gently dipped in 2-4% is without calcium ion sodium alginate soln with tweezers, so
It is neatly placed on afterwards in the metal net shaped carrier strips of 5mm × 20mm, 5-10 stem apex in every carrier strip.Subsequently by the dress with stem apex
Carrier strip is immersed containing 0.1-0.3% calcium chloride, 0.4 molL-1Sucrose and 2moL-1The MS fluid nutrient mediums of glycerine, wherein, contain
0.1-0.3% calcium chloride, 0.4 molL-1Sucrose and 2moL-1Without NH4+ in the MS fluid nutrient mediums of glycerine, consolidate through 10-30min
After fixed, carrier strip is sucked into surplus liquid on aseptic filter paper.The carrier strip with stem apex fixed is put in preprocessing solution,
The preprocessing solution contains 0.4 molL-1And glycerine 2moL-1MS fluid nutrient mediums, without being handled at room temperature in NH4+
After 20-60min, the ice bath processing carried out in vitrifying protective agent PVS2 2-5 hours is transferred to, NH4 is free of in the PVS2+。
Carrier strip with stem apex is put into liquid nitrogen rapidly and is preserved.
The present invention also provides a kind of renewal cultivation methods after wild rice stem apex cryopreservation, include the following steps：
Wild rice stem apex after cryopreservation takes out the carrier strip with stem apex when thawing from liquid nitrogen, is immediately placed in
1.2molL-1, without NH4+1/2MS fluid nutrient mediums in quick-thawing 2min, and wash 20min in the medium.By band
The carrier strip of stem apex blots on filter paper to be placed on without hormone and without NH4+1/2MS culture mediums in light culture 1 day, then
Stem apex by carrier strip is removed one by one under anatomical lens, is transferred to containing BA and NAA, without NH4+Culture medium on secretly trained
It supports.It is transferred to after stem apex regeneration containing BA and NAA and without NH4+Culture medium illumination cultivation.
The invention has the advantages that the cryopreservation method of wild rice stem apex is simple and easy to do, and it is reliable and stable, after preservation
Wild rice restoration ecosystem is in order.Meanwhile by adding exogenous hormone and stepping up both sucrose concentrations in pre-culture medium
With reference to the resistance for improving stem apex, by using metal grill as the carrier of stem apex to reduce the damage in each step, and use
Liquid nitrogen is delivered directly and the freezing step of non-dependent programmed cooling instrument, and a great number of elements is adjusted in recovery media and is allowed to easy
Regeneration finally obtains the regeneration plant of common wild-rice.Other types of wild rice and grass genetic resources are surpassed
For Cord blood, there is good reference value.The resistance of stem apex is also improved simultaneously, reduces the damage in each step, and adopt
It is delivered directly and the freezing step of non-dependent programmed cooling instrument with liquid nitrogen, simplifies operation, establish the cryopreservation body of common wild-rice
System, and made technological reserve for other types of wild rice and grass.
Description of the drawings
Fig. 1 is the flow chart of wild rice cryopreservation in the present invention.
Fig. 2 is the flow chart of renewal cultivation method after wild rice cryopreservation in the present invention.
Fig. 3 is the status diagram of tufted seedling in the present invention.
Fig. 4 is the status diagram of domesticated seedlings in the present invention.
Fig. 5 is the status diagram of embedding process in the present invention.
Fig. 6 is the status diagram of regrowth in the present invention.
Specific embodiment
In order to more clearly describe the specific embodiment of the present invention, the present invention is done further with reference to embodiment
Explanation.
The acquisition of 1 wild rice aseptic seedling of embodiment
With reference to shown in Fig. 1, the induction of wild rice aseptic seedling and subculture step are：The subterranean stem band of wild rice is taken to germinate
The stem section of bud, after rinsing about 1 hour repeatedly under tap water flowing water, with 70% alcohol disinfecting, 1 min, 0.1% mercuric chloride disinfection 15
Min, aseptic water washing several times after, the stem apex of about 2mm is stripped under anatomical lens, is inoculated in containing 4 mgL-1BA and 0.5
mg·L-1Multiple Buds are induced on the MS culture mediums of NAA.Cultivation temperature is 25 ± 2 DEG C, 12 hd of illumination-1, intensity of illumination 36
µmolm-2 s-1.After inducing Multiple Buds, Multiple Buds are cut off and are seeded in containing 2mgL-1BA and 0.5 mgL-1The MS of NAA
Squamous subculture on culture medium.Every 3 months subcultures are primary.
The gradient preculture of 2 wild rice of embodiment
With reference to shown in Fig. 1, the gradient of aseptic seedling, which tames the step of cultivating, removing stem apex and stem apex preculture, is：Take implementation
The tissue-cultured seedling of 1 gained of example, cuts off top plant, base portion about 3cm, root about 1cm is stayed to be put into the MS medium cultures 7 without hormone
After it, base portion is taken newly to sprout the bud of 1cm, be transferred to 0.3molL containing sucrose-1And abscisic acid 5mgL-1Culture medium on cultivate.After 7 days,
It is transferred to 0.7molL again-1And abscisic acid 5mgL-1Culture medium on preculture 5 days.The stem apex of 2-3mm is stripped under anatomical lens, containing
There is 0.4 molL-1And glycerine 2moL-1, without NH4+MS solid mediums on preculture 16 hours.
The device of 3 wild rice stem apex of embodiment is fixed
It is with reference to the step of shown in Fig. 1 and Fig. 5, stem apex is fixed to carrier strip and pretreatment, vitrifying processing：It will with tweezers
By the stem apex after 2 preculture of embodiment, gently dipped in 2-4% is without calcium ion sodium alginate soln, i.e., be neatly placed on 5mm ×
In the metal net shaped carrier strips of 20mm, 10 stem apexs in every carrier strip.By with stem apex carrier strip immerse containing 0.2% calcium chloride,
0.4 molL-1Sucrose and 2moL-1Glycerine, without NH4+MS fluid nutrient mediums in, after 20min is fixed, by carrier strip in nothing
Surplus liquid is sucked on bacterium filter paper.
The vitrifying of 4 wild rice of embodiment is handled and is frozen
With reference to shown in Fig. 1 and Fig. 5, the step of Liquid nitrogen of stem apex, is：Example 3 gained with stem apex
Carrier strip is put in preprocessing solution, contains 0.4 molL-1And glycerine 2moL-1, without NH4+MS fluid nutrient mediums in room temperature
After lower processing 60min, vitrifying protective agent PVS2 is transferred to, without NH4+The middle ice bath processing for carrying out 5 hours, by the dress with stem apex
Carrier strip puts into liquid nitrogen preserve rapidly.
The stem apex renewal cultivation and plant regeneration of 5 wild rice of embodiment
With reference to shown in Fig. 2, Fig. 3, Fig. 4, Fig. 6, the required step of renewal cultivation method is the first step：Rapidly thaw；Second
Step：It washs, blot；Third walks, stem apex renewal cultivation；4th step, stem apex are removed by carrier strip；5th step：Stem apex regeneration culture,
6th step：Tissue culture plant regenerates.
Specific steps are as follows：The carrier strip with stem apex is taken out during defrosting from liquid nitrogen, is immediately placed in 1.2molL-1, be free of
NH4+1/2MS fluid nutrient mediums in quick-thawing 2min, and wash 20min in the medium.Carrier strip with stem apex is existed
It blots and is placed on without hormone on filter paper, light culture 1 day in the 1/2MS culture mediums without NH4+, then by stem under anatomical lens
Point is removed one by one by carrier strip, is transferred to containing BA and NAA, without NH4+Culture medium on light culture.It is transferred to after stem apex regeneration
Containing BA and NAA, containing NH4+Culture medium illumination cultivation.After stem apex renewal cultivation 3d, Microscopic observation is trained in solution, you can seen stem
Point regeneration, after continuing culture about 14~28 days, partly survives stem apex brown stain, partial regeneration Cheng Lvmiao, regeneration rate reaches as high as
50%。
Embodiment described above only expresses embodiments of the present invention, and description is more detailed, as long as the skill of this field
Art personnel are after the embodiment of the present invention is viewed, and under the premise of not departing from present inventive concept, the change made belongs to this hair
Bright protection domain.But embodiment as described herein is it is not intended that limit protection scope of the present invention.
Claims (2)
1. a kind of cryopreservation method of wild rice stem apex, which is characterized in that the store method includes the following steps：
Step a:The sterile tissue-cultured seedling of squamous subculture 3 months or more is taken, top plant is cut off, base portion is stayed to be put into the MS without hormone
It is cultivated in culture medium, the sterile tissue-cultured seedling of subculture in the step a 3 months or more cuts off top plant, stays base portion about
3cm, root about 1cm are put into the MS culture mediums without hormone；
Step b:After being cultivated 5-7 days by step a, base portion is newly sprouted after plant cuts off, be transferred to base portion containing 0.3molL-1's
Sucrose and abscisic acid 0.5-5mgL-1Culture medium on cultivate, the length of the new rudiment of base portion in the step b is 0.5-1cm；
Step c:Plant after step b is cultivated 7-10 days, then it is transferred to 0.7molL-1Sucrose and 0.5-5mg containing abscisic acid
L-1Culture medium on preculture 3-7 days；
Step d:Plant after step c precultures 3-7 days strips the stem apex of 2-3mm under anatomical lens, is containing 0.4
mol·L-1Sucrose and glycerine 2molL-1MS solid mediums on preculture 16 hours, be free of in the MS solid mediums
NH4 +；
Step e:The stem apex after step d precultures is gently dipped in 2-4% is without calcium ion sodium alginate soln with tweezers,
Then it is neatly placed in the metal net shaped carrier strips of 5mm × 20mm, 5-10 stem apex in every carrier strip；
Step f:Carrier strip with stem apex is immersed containing 0.1-0.3% calcium chloride, 0.4 molL-1Sucrose and 2molL-1It is sweet
In the MS fluid nutrient mediums of oil, after 10-30min is fixed, carrier strip is sucked into surplus liquid, the MS liquid on aseptic filter paper
NH is free of in body culture medium4 +,
Step g:The carrier strip with stem apex fixed is put in after handling 20-60min at room temperature in preprocessing solution, be transferred to
The ice bath processing of 2-5 hours is carried out in vitrifying protective agent PVS2, the carrier strip with stem apex is put into liquid nitrogen rapidly and is preserved,
In, the preprocessing solution contains 0.4 molL-1Sucrose and glycerine 2molL-1, without NH4 +MS fluid nutrient mediums, institute
Vitrifying protective agent PVS2 is stated without NH4 +。
2. a kind of renewal cultivation method after wild rice cryopreservation, which is characterized in that the wild rice is protected by ultralow temperature
The method of depositing preserved, and takes out the carrier strip with stem apex during defrosting from liquid nitrogen, is immediately placed in 1.2molL-11/2MS liquid training
Quick-thawing 2min in base is supported, and washs 20min in the medium, the 1.2molL-11/2MS fluid nutrient mediums be free of
NH4 +, the carrier strip with stem apex on filter paper is blotted and is placed on light culture 1 day, Ran Hou in the 1/2MS culture mediums without hormone
Stem apex by carrier strip is removed one by one under anatomical lens, is transferred on the culture medium containing BA and NAA and carries out light culture, it is described containing BA and
The culture medium of NAA is free of NH4 +, the culture medium illumination cultivation containing BA and NAA, the stem are transferred to after the regeneration of processed stem apex
The culture medium containing BA and NAA is transferred to after point regeneration containing NH4 +。
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| CN109601378B (en) * | 2018-11-30 | 2021-08-24 | 广东省生物工程研究所（广州甘蔗糖业研究所） | Low-temperature preservation method and cultivation method for seed source of sugarcane tissue culture seedling |
| 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 |
| CN115843687B (en) * | 2022-12-06 | 2023-09-01 | 上饶师范学院 | Method for promoting expansion of test tube taro through embedded vitrification ultralow temperature therapy detoxification of red bud taro |
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