CN106993609A - A kind of spindle tree stem apex cryopreservation method - Google Patents
A kind of spindle tree stem apex cryopreservation method Download PDFInfo
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- CN106993609A CN106993609A CN201710239317.4A CN201710239317A CN106993609A CN 106993609 A CN106993609 A CN 106993609A CN 201710239317 A CN201710239317 A CN 201710239317A CN 106993609 A CN106993609 A CN 106993609A
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- stem apex
- spindle tree
- cryopreservation method
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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
Abstract
The present invention relates to a kind of spindle tree stem apex cryopreservation method, include successively：The stripping of spindle tree stem apex, stem apex pretreatment, loading, PVS2 vitrifyings processing, Liquid nitrogen storage, the method for defrosting and unloading and renewal cultivation.The present invention have it is simple to operate, occupy little space, safety and stability, preserve low cost and other advantages, while the stem apex after cryopreservation can preserve its genetic integrity and stability.In addition low temperature environment can also avoid the introducing of pest and disease damage, in terms of the spindle tree crossbreeding and molecular studies on there is important practice and dissemination.
Description
Technical field
The present invention relates to a kind of stem apex long-term preservation method, specifically a kind of spindle tree stem apex cryopreservation method.
Background technology
Spindle tree, also known as white Du, winterberry euonymus herb, are a kind of dungarungas of Celastraceae Euonymus, being that China is common views and admires
One of class flower garden seeds, its is leaf beautiful, and plant is in great numbers, is coated with the fruit of red aril and can harbor for a long time in end of the branch, arrives
Winter, white snow haw sets each other off, very beautiful, is the northern gardens in winter add many colors.Spindle tree is cold-resistant, anti-
There is good adaptability in terms of drought, wind resistance；Spindle tree fruit total saposins are to Bel7402-SMMC, human cervical carcinoma
Cell line-HeLa and breast cancer cell line-MCF7 have obvious suppression growth；The seed of spindle tree is containing abundant fat
Class material, oil content is up to more than 40%, can be used as iundustrial oil；Timber exquisiteness is tough and tensile, available for carving, boom processed or coaster
Deng；Seed and root are medicinal, available for diseases such as the treatment pain of the knee joint, dermatitis rhus.As can be seen here, spindle tree be a kind of collection industry,
It is medicinal, view and admire applied to one plant, with higher promotional value.Preserved according to traditional plantation, easily make its by
To germ or the influence of insect pest, the loss of great manpower financial capacity is in turn resulted in, because stem apex has good meristematic capacity, because
This has widely been applied to the Plantlet in vitro of plant germplasm resource at present.
Plant germplasm resource cryopreservation refer to by plant cell, tissue or organ liquid nitrogen (- 196 DEG C) ultralow temperature
Under the conditions of preserved.At such lower temperatures, participated in vegetable material metabolism various biology enzymes activity by
Greatly suppress, organism metabolism is basic to be stopped, and in " seemingly-dead " state, plant germplasm resource can be realized using this method
Persistence.Successfully carrying out the material type of cryopreservation at present has seed, resting bud, shoot apical meristem, pollen, conjunction
Sub- embryo (plumular axis), somatic embryo, suspension cell, callus, protoplast etc..And compared with other positions of plant, top point
Raw tissue has a stronger meristematic capacity, and when stem apex can still keep stronger meristematic capacity after cryopreservation,
Therefore stem apex is also one of preferred material of cryopreservation, therefore has been widely used for plant germplasm resource ultralow temperature guarantor
Deposit.
Cryopreservation technology is preserved applied to stem apex, its advantage：Operation is simple, occupy little space, safety steady
Surely preserve cost low while breeding work can also be served very well, the introducing of pest and disease damage can also be avoided, be interzone, international
Kind mass transter provider just.
Spindle tree stem apex cryopreservation yet there are no report.Therefore, research and development one kind is suitable for spindle tree stem apex
Feasible cryopreservation method be very important.
The content of the invention
The purpose of the present invention is to be achieved through the following technical solutions：
The present invention relates to a kind of method that spindle tree stem apex vitrification ultra-low temperature is preserved, include successively：Spindle tree stem
Point stripping, stem apex pretreatment, loading, PVS2 vitrifyings processing, Liquid nitrogen storage, defrosting and unload and renewal cultivation side
Method.
Preferably, the stripping of described spindle tree stem apex is specifically referred to：Aseptically, from the sterile of healthy growth
The stem apex length stripped on spindle tree tissue-cultured seedling is 2~3mm Shoot Tips.
Preferably, described pretreatment refers to：Stem apex after stripping is put into pre-culture solution and handled, sugarcane in pre-culture solution
Sugared optimum concentration is 0.4mol/L MS salting liquids, the light culture 2d in environmental condition is 25 DEG C of growth cabinet.
Preferably, described loading is specifically referred to：Shoot Tips after preculture are transferred in loading liquid, at room temperature
Load 10~30min；The composition of described loading liquid is：The MS salting liquids of 2M glycerine+0.4M sucrose.
Preferably, described vitrifying processing is specifically referred to：Laden stem apex is put into 100%PVS2 vitrification solutions
30min is handled under the conditions of 0 DEG C；The composition of described PVS2 vitrification solutions is：The second two of+30% glycerine of MS salting liquids+15%
Alcohol 15%+15% dimethyl sulfoxide (DMSO)+0.4M sucrose.
Preferably, described Liquid nitrogen storage refers to：Stem apex after the processing of PVS2 solution is changed into new PVS2 solution, then
It is rapid to immerse cryovial in liquid nitrogen, at least freeze more than 1h.
Preferably, described defrosting and unloading refer to：Cryovial after Liquid nitrogen storage is taken out and changed in 40 DEG C of water-baths
Freeze 70s, then cryovial is gone on superclean bench wash 20min with unloading carrier fluid；The described component for unloading carrier fluid is：MS salt is molten
Liquid+1.2M sucrose.
Preferably, described renewal cultivation is specifically referred to：The stem apex unloaded after carrier fluid washing is seeded on recovery media
Carry out renewal cultivation；Described recovery media is MS+6-BA1.0mg/L；Described renewal cultivation condition is：First in 25 DEG C of people
Light culture 7d in work climate box, is then transferred to tissue-cultured seedling culturing room and is cultivated.
Compared with prior art, there are following beneficial outcomes in the present invention：
A kind of method of spindle tree stem apex cryopreservation of the present invention is a kind of long-term safety, reliable and stable, simple
Spindle tree stem apex survival rate is up to 70% after the effective method for preserving spindle tree fine germplasm resources, cryopreservation；
The present invention is using adventitious bud stem apex as cryopreservation material, one side convenient material drawing, on the other hand because stem apex contains stem apex point
Raw tissue, cytothesis ability is stronger, and genetic stability is high.
Brief description of the drawings
Fig. 1 is influence of the spindle tree stem apex length to stem apex survival rate after cryopreservation in the embodiment of the present invention 3
Fig. 2 is influence of the pre-culture solution sucrose concentration to stem apex survival rate after cryopreservation in the embodiment of the present invention 4
Fig. 3 is influence of the pre-incubation time to stem apex survival rate after cryopreservation in the embodiment of the present invention 4
Fig. 4 is influence of the loading liquid processing time to stem apex survival rate after cryopreservation in the embodiment of the present invention 5
Fig. 5 is influence of the PVS2 solution processing times to stem apex survival rate after cryopreservation in the embodiment of the present invention 6
Fig. 6 be the embodiment of the present invention 7 in unload influence of the carrier fluid processing time to stem apex survival rate after cryopreservation
Fig. 7 is spindle tree stem apex cryopreservation system process figure in the present invention；A spindle tree seedling terminal buds；B peaches
Leaf winged euonymus Shoot Tips；The spindle tree stem apex of recovery media is transferred to after c cryopreservations；Spindle tree after d light cultures 7d
Stem apex survival condition；The stem apex survived after e cryopreservations；Dead stem apex after f cryopreservations；After g, h cryopreservation
The callus of spindle tree stem apex formation；The propagation of spindle tree stem apex callus after i cryopreservations；J ultralow temperature is protected
Deposit the differentiation of rear spindle tree stem apex callus；The propagation of k Bud Differentiations；L Bud Differentiation seedlings；Peach leaf is defended after m cryopreservations
Lance regrowth is taken root；The transplanting of spindle tree regrowth after n cryopreservations.
Embodiment：
The present invention is described in further details with reference to embodiment, the present embodiment using technical solution of the present invention before
Put and implemented, give detailed embodiment and specific operating process.
Embodiment 1
A kind of method of spindle tree stem apex cryopreservation, concrete operations are as follows：
1) the stem apex length aseptically, stripped from the sterile spindle tree tissue-cultured seedling of healthy growth is 2~3mm
Shoot Tips；
2) stem apex after stripping is put into pre-culture solution and handled, sucrose optimum concentration is 0.4mol/L's in pre-culture solution
MS salting liquids, the light culture 2d in environmental condition is 25 DEG C of growth cabinet；
3) Shoot Tips after preculture are transferred in loading liquid, 10~30min is loaded at room temperature；Described loading
The composition of liquid is：MS salting liquid+2M glycerine+0.4M sucrose；
4) laden stem apex is put into 100%PVS2 vitrification solutions under the conditions of 0 DEG C and handles 30min；Described
The composition of PVS2 vitrification solutions is：Dimethyl sulfoxide (DMSO)+0.4M the sucrose of+15% ethylene glycol of+30% glycerine of MS salting liquids+15%；
5) stem apex after the processing of PVS2 solution is changed into new PVS2 solution, it is then rapid to immerse cryovial in liquid nitrogen,
At least freeze more than 1h；
6) cryovial after Liquid nitrogen storage is taken out and the 70s that thawed in 40 DEG C of water-baths, then cryovial is gone into ultra-clean work
Make on platform to wash 20min with unloading carrier fluid；The described component for unloading carrier fluid is：MS salting liquid+1.2M sucrose；
7) stem apex unloaded after carrier fluid washing is seeded on recovery media and carries out renewal cultivation；Described recovery media
For MS+6-BA1.0mg/L；Described renewal cultivation condition is：First the light culture 7d in 25 DEG C of growth cabinets, is then transferred to group
Pei Miao culturing room is cultivated.
The acquisition of the aseptic seedling of embodiment 2
In the vegetative growth phase (5~August part) of spindle tree, the explant with stem apex is taken back into use for laboratory liquid detergent water
Cleaned, and 1~2h of flushing under circulating water.Explant is placed on superclean bench and sterilized by flushing after finishing.
First with 75% alcohol to its surface sterilizing 10s, with aseptic water washing 3~4 times；When recycling different bactericidal agent processing different
Between carry out depth sterilizing (as shown in table 1), finally with aseptic water washing 6~8 times.Then the stem apex of stripping is inoculated in MS+6-
BA2.0mg/L+NAA0.5mg/L+30g/L sucrose+7g/L agar, pH value is on 5.8 culture medium.Counted after 15d in each bottle
Pollution rate, the death rate and survival rate.Simultaneously in order to study the optimum culture medium of callus, stem apex is inoculated in difference
On concentration cultures, using MS as minimal medium, add to count after 30g/L sucrose, the 6-BA and NAA, 30d of various concentrations and be cured
Injured tissue inductivity and its growing state.Then using the callus of gained as material, to study the differentiation of optimum callus
The hormone in medium proportioning of young shoot, is added after being combined the 6-BA and NAA of various concentrations in MS minimal mediums, other
Condition is set to 30g/L sucrose+7g/L agar, and pH value is 5.8.Then it is peeled off when Bud Differentiation length is to more than 3cm
It is inoculated on 1/2MS+NAA0.2mg/L root media.
Result of study shows that (as shown in table 2) uses HgCl2The germination rate of explant will be apparently higher than use after being sterilized
NaClO is sterilized, and pollution rate is small.And with the increase of processing time, explant pollution rate is being gradually reduced, but its
The death rate is also in increase, it has also been found that part stem apex explant is not due to by HgCl in specific test operation2Rinse well, explant
There is the withered phenomenon of jaundice, therefore 75% alcohol 10s+0.1%HgCl of selection in body24min is used as optimal sterilizing methods.In addition
Induction of 6-BA and the NAA combination of various concentrations to spindle tree callus plays obvious action (as shown in table 3), with
6-BA concentration is gradually stepped up, and the healing rate of spindle tree callus shows the trend for first raising and reducing afterwards；As NAA is dense
That spends gradually rises, and the healing rate of spindle tree callus shows the trend gradually reduced.In addition as 6-BA concentration and NAA
When concentration difference is larger, healing rate is high.Finally compare and understand when 6-BA concentration is 1.0mg/L, and NAA concentration is 0.1mg/L, peach
Leaf winged euonymus healing rate reaches as high as 98.0%, and growing state is good.Break up the result of study of optimum medium simultaneously for bud
Show that the differentiation and propagation of the 6-BA and NAA of various concentrations to bud have obvious effect (as shown in table 4).With 6-BA concentration
Gradually rise, the differentiation rate and growth coefficient of bud are continuously increased on callus；And when 6-BA is when NAA is combined, bud
Different changes are presented in differentiation rate.When 6-BA concentration is 0.5mg/L, no matter the height of NAA concentration, the differentiation rate and propagation of bud
Coefficient is all than relatively low, and the growing way of Bud Differentiation is weak；When callus is seeded in 6-BA concentration for 1.5mg/L, NAA concentration is
When on 0.2mg/L culture medium, the differentiation rate of bud is up to 99%, and value-added coefficient is 3.21, and the upgrowth situation of Bud Differentiation is good.
Table 1 tests different sterilization methods combination used
The influence that the different bactericidal agents of table 2 and its processing time grow to spindle tree explant
The influence of table 3 different growth regulators and its concentration proportioning to callus induction
The different growth regulators of table 4 and its concentration proportioning break up the influence with breeding to bud
Embodiment 3
Test influence of the stem apex length to spindle tree stem apex cryopreservation：Healthy growth is stripped under aseptic condition
The stem apex of sterile spindle tree tissue-cultured seedling, by stereomicroscope it is observed that spindle tree stem apex position is symmetric form, because
This can determine stem apex length in stripping process according to the logarithm of remaining spire, set 1~2mm (not include phyllogen altogether
Base), 2~3mm (comprising a pair of phyllopodium), 3~5mm (including two pairs of phyllopodium), three gradients.Result of study shows (such as Fig. 1
It is shown) spindle tree stem apex length has a significant impact to stem apex survival rate after cryopreservation.When stem apex length is 2~3mm,
Stem apex survival rate is up to 83.33% after cryopreservation, much larger than the stem apex that length is 1~2mm and 3~5mm.When stem apex length
During for 1~2mm, because no spire coating causes separate living tissue to bear larger infringement outside shoot apical meristem, final inactivation；
And when stem apex length is 3~5mm, although shoot apical meristem obtains good protection, but is due to that stem apex volume is excessive, leads
Cause the shoot apical meristem position dehydration in During Vitrification in vitro not thorough, and then to form crystallization in Cryopreservation
Eucaryotic cell structure is destroyed, finally causes stem apex inactivation.Therefore select appropriate to cut length for ultralow in stem apex stripping process
It is vital that stem apex, which is survived, after temperature is preserved.
Embodiment 4
Test the influence of sucrose concentration and pre-incubation time to spindle tree stem apex cryopreservation in pre-culture medium：Only
Change the sucrose concentration and pre-incubation time in pre-culture medium, the stem apex stripped is respectively put into equipped with various concentrations first
Preculture is carried out in the MS salting liquids of (0.1M~0.5M) sucrose；Preculture environmental condition is 25 DEG C of light cultures, and incubation time is
(0、1d、2d、3d)；Then the survival rate of stem apex renewal cultivation after cryopreservation is counted.Result of study shows (such as Fig. 2
Shown in Fig. 3) stem apex of stripping is put into the pre-culture solution of different sucrose after progress preculture different time, stem apex passes through
Survival rate after cryopreservation is had nothing in common with each other, and when sucrose concentration is 0.4M, when pre-incubation time is 2d, stem apex survival rate is most
It is high by reachable 60%.It can thus be appreciated that preculture is to influence the key factor of spindle tree stem apex cryopreservation survival rate, optimal
Preculture sucrose concentration is 0.4M, and incubation time is 2d.
Embodiment 5
Test influence of the loading liquid processing time to stem apex survival rate after cryopreservation：Stem apex is handled by preculture
After be transferred to superclean bench, stem apex is together poured into culture dish (d=120mm) together with pre-culture solution, then with sterile toothpick will
Stem apex is chosen in the cryovial equipped with Loading solution, load at room temperature different time (0,10,20,30min).Research knot
Fruit shows (as shown in Figure 4) stem apex by there is significant difference between loading liquid processing and control group, when stem apex is without loading liquid
When processing directly carries out PVS2 dewater treatments, stem apex survival rate is only 6%；When loading liquid processing time be 10min when, stem apex into
Motility rate handles 10min survival rate and processing 20min, 40min up to 70%, and with the further extension of loading time
As a result without significant difference, therefore preservation efficiency is improved in order to save the time, the loading liquid optimization process time is 10min.
Embodiment 6
Test influence of the PVS2 solution processing times to stem apex survival rate after cryopreservation：Stem apex passes through Loading
Be transferred to after solution processing in vitrification solution PVS2, handled in 0 DEG C of ice chest different time (0,20,40,60min) after,
Determine the survival rate of stem apex；Result of study shows (as shown in Figure 5) when stem apex is without in PVS2 solution processing direct plunge into Liquid Nitrogen
After preservation, stem apex is all inactivated in renewal cultivation to bleach；When PVS2 processing times are 60min, stem apex survival rate highest can
Up to 73.3%, and when treated between when further extending to 80min, survival rate is reduced to 50%.
Embodiment 7
Test the influence for unloading carrier fluid processing time to stem apex survival rate after cryopreservation：It will be equipped with the cryovial of stem apex
The 70s that thawed in 40 DEG C of water-baths is put into after being pulled out from liquid nitrogen rapidly, then the stem apex in cryovial is transferred to Uploading solution
Middle processing different time (0,10,20,30min), washing process is carried out in gnotobasis；Result of study shows (such as Fig. 6 institutes
Show) when directly carrying out renewal cultivation without the stem apex for unloading carrier fluid processing, stem apex is all dead, illustrates still there is residual outside stem apex
The PVS2 solution stayed；Using the MS salting liquids of the sucrose containing 1.2M to thawing after stem apex washing 10min after, stem apex survival rate is reachable
More than 80%, the influence of the length of wash time to stem apex survival rate is not notable.
Embodiment 8
Test influence of the recovery media to stem apex survival rate after cryopreservation：Spindle tree after cryopreservation
After stem apex is washed through Uploading solution, it is seeded on different recovery medias and is cultivated, condition of culture is first at 25 DEG C
Light culture 7d in growth cabinet, is then transferred to tissue-cultured seedling culturing room and is cultivated.Will according to the proportioning of different growth regulators
Recovery media is set to：①MS；②MS+6-BA1.0mg/L；③MS+6-BA1.0mg/L+NAA0.1mg/L；Result of study table
Culture medium of survival rate of bright (as shown in table 5) stem apex in the culture medium without hormone less than addition hormone；Additionally may be used
To find out recovery media of the stem apex recovery effects better than addition 6-BA and NAA in only addition 6-BA recovery media, by
This understands that 6-BA has for stem apex survival rate after cryopreservation to be significantly affected.
Influence of the recovery media of table 5 to stem apex survival rate after cryopreservation
The above embodiments merely illustrate the technical concept and features of the present invention, and its object is to allow be familiar with this art
Personage can understand present invention and be carried out, and it is not intended to limit the scope of the present invention.
Claims (8)
1. a kind of spindle tree stem apex cryopreservation method, it is characterised in that include successively：The stripping of spindle tree stem apex,
Stem apex pretreatment, loading, PVS2 vitrifyings processing, Liquid nitrogen storage, defrosting and unloading and renewal cultivation method.
2. spindle tree stem apex cryopreservation method according to claim 1, it is characterised in that：Described spindle tree
The stripping of stem apex is specifically referred to：Aseptically, the stem apex stripped from the sterile spindle tree tissue-cultured seedling of healthy growth is long
Spend the Shoot Tips for 2~3mm.
3. spindle tree stem apex cryopreservation method according to claim 1, it is characterised in that：Described pretreatment is
Refer to the stem apex after stripping being put into pre-culture solution and handle, sucrose optimum concentration is molten for 0.4mol/L MS salt in pre-culture solution
Liquid, the light culture 2d in environmental condition is 25 DEG C of growth cabinet.
4. spindle tree stem apex cryopreservation method according to claim 1, it is characterised in that：Described loading is specific
Refer to transfer the Shoot Tips after preculture in loading liquid, 10~30min is loaded at room temperature；The group of described loading liquid
Turn into：MS salting liquid+2M glycerine+0.4M sucrose.
5. spindle tree stem apex cryopreservation method according to claim 1, it is characterised in that at described vitrifying
Reason is specifically referred to：Laden stem apex is put into 100%PVS2 vitrification solutions under the conditions of 0 DEG C and handles 30min；Described
The composition of PVS2 vitrification solutions is：Dimethyl sulfoxide (DMSO)+0.4M the sucrose of+15% ethylene glycol of+30% glycerine of MS salting liquids+15%.
6. spindle tree stem apex cryopreservation method according to claim 1, it is characterised in that described Liquid nitrogen storage
Refer to：Stem apex after the processing of PVS2 solution is changed into new PVS2 solution, it is then rapid to immerse cryovial in liquid nitrogen, it is at least cold
Freeze more than 1h.
7. spindle tree stem apex cryopreservation method according to claim 1, it is characterised in that described defrosting and unload
Load refers to：Cryovial after Liquid nitrogen storage is taken out and the 70s that thawed in 40 DEG C of water-baths, then cryovial is gone into superclean bench
It is upper to wash 20min with unloading carrier fluid；The described component for unloading carrier fluid is：MS salting liquid+1.2M sucrose.
8. spindle tree stem apex cryopreservation method according to claim 1, it is characterised in that：Described renewal cultivation
Specifically refer to the stem apex unloaded after carrier fluid washing being seeded on recovery media and carry out renewal cultivation；Described recovery media is
MS+6-BA1.0mg/L；Described renewal cultivation condition is：First the light culture 7d in 25 DEG C of growth cabinets, is then transferred to tissue culture
Seedling culturing room is cultivated.
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