CN102640745B - Ultralow-temperature cryopreservation and recovery method for embryonic materials - Google Patents
Ultralow-temperature cryopreservation and recovery method for embryonic materials Download PDFInfo
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- CN102640745B CN102640745B CN201210135123.7A CN201210135123A CN102640745B CN 102640745 B CN102640745 B CN 102640745B CN 201210135123 A CN201210135123 A CN 201210135123A CN 102640745 B CN102640745 B CN 102640745B
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Abstract
The invention discloses an ultralow-temperature cryopreservation and recovery method for embryonic materials, which includes inoculating hypertonic liquid medium to embryonic material to be preserved, and culturing at 25 DEG C for 3-10 days; filtering the embryonic material and loading the same into a freeze tube, adding composite vitrified protectant, dewatering at 0 DEG C for 0-70 minutes, placing the material into a freezing box, directly placing the box into liquid nitrogen for quick freezing, and preserving with the liquid nitrogen for long term; and recovering the stored material by washing, hypertonic culture, hypotonic culture and conventional culture sequentially. The ultralow-temperature cryopreservation and recovery method for embryonic materials is simple to operate, convenient and feasible. By the method, the important bottleneck in industrialization of tree somatic embryos is broken through, the embryonic materials can be stored for over one year, and high-frequency proliferation of the materials can be kept. The method is highly practical, and great economic benefit and social benefit can be achieved.
Description
Technical field
The present invention relates to the store method of embryo material in forest genetics, the superfreeze that is specifically related to an embryo material is preserved method for resuscitation.
Background technology
Forest somatic embryo occur and plant regeneration system in, whether the cells,primordial in culture materials can keep vigorous point to be also the important bottleneck of forest body embryo industrialization with Regeneration Ability the long period.At present, also do not have preferably for preserving the method for embryo material, conventional is exactly low-temperature preservation, although this method is simple, the holding time is short, and cellular-restoring difficulty, and multiplication capacity is low, can not preserve for a long time at all.
Summary of the invention
Goal of the invention: for the deficiencies in the prior art, the superfreeze that the object of this invention is to provide an embryo material is preserved method for resuscitation, preserves and can make it keep high frequency multiplication capacity to realize embryo material.
Technical scheme: in order to realize foregoing invention object, the technical solution used in the present invention is as follows:
The superfreeze of one embryo material is preserved method for resuscitation, comprises the following steps:
(1) get embryo material to be preserved, by 10% inoculum concentration access hypertonic liquid medium, 25 ℃, cultivate 3 ~ 10d; Wherein, hypertonic liquid medium is for being added with the M13 minimal medium of 0.1 ~ 0.8mg/L sorbierite;
(2) the pre-incubated embryo material of filtration step (1), packs cryovial into, in cryovial, adds compound glass protectant, and 0 ℃, place dehydration 0～70min, then pack the quick-frozen of freeze box direct plunge into Liquid Nitrogen into, the medium-term and long-term preservation of liquid nitrogen; Compound glass protectant is the M13 minimal medium that adds 30% glycerine and 30% ethylene glycol;
(3) from liquid nitrogen, take out freeze box, rapidly cryovial is placed in to 35 ~ 43 ℃ of water-baths, 2min fast thaws; Suck compound glass protectant, add the height of 1mL to ooze callus proliferated culture medium to cryovial, stop 2min, for several times repeated washing; Height oozes callus proliferated culture medium for adding the M13 minimal medium of 0.1 ~ 0.5M sucrose;
(4) the embryo material after washing is transferred to rapidly to the height that is lined with filter paper and ooze callus proliferated culture medium, 25 ℃, cultivate 1 ~ 6d;
(5) turn embryo material and cultivate 1 ~ 6d to hypotonic callus proliferated culture medium, grow new callus, then transfer on callus proliferated culture medium and cultivate; Hypotonic callus proliferated culture medium is for adding the M13 minimal medium of 0.1 ~ 0.4M sucrose;
(6) shifting length has the embryo material of new callus to cultivate to body embryonal induction medium, until high frequency somatic embryo occurs.
In step (1), cultivate 6 ~ 8d.
In step (1), in described hypertonic liquid medium, be also added with 1 ~ 5mg/L ABA and 50 ~ 200mg/L proline.
In step (2), described placement dehydration,, then forwards 100% PVS dehydration 0 ~ 60min to, and then drops into liquid nitrogen and preserve with 60% PVS2 protection embryo material transition dehydration 0 ~ 10min for first.
In step (3), 37 ℃ of water-baths.
In step (3), it is the M13 minimal medium that adds 0.6mol/L sorbierite that height oozes callus proliferated culture medium.
In step (4), sucrose concentration is 0.4M, cultivates 2d.
In step (5), sucrose concentration is 0.3M, cultivates 5d.
In step (6), cultivate 3 months, just can realize high frequency somatic embryo and occur.
Beneficial effect: compared with prior art, the superfreeze of embryo material of the present invention is preserved method for resuscitation, simple to operate, facilitate feasible, the important bottleneck problem that has solved the industrialization of forest body embryo, can realize embryo material and be saved in more than 2 years, and can make it keep high frequency multiplication capacity, there is good practicality, can produce good economic benefit and social effect.
Accompanying drawing explanation
Fig. 1 is preculture bleeding agent concentration result of the test figure;
Fig. 2 is preculture time result of the test figure;
Fig. 3 is that different temperatures is carried out cell recovery result figure;
Fig. 4 is embryonal suspension cell recovery result figure;
Fig. 5 is the exercising result figure of soluble starch in renewal cultivation; In figure, A, B are the state while recovering 5d, and C, D are the state while recovering 25d;
What Fig. 6 was ABA on cell survival affects result figure;
Fig. 7 is the result of variations figure of endogenous proline after sorbierite is processed;
Fig. 8 is the protective effect result figure of Foreign Proline to cell.
Embodiment
Below in conjunction with specific embodiment, the present invention is further illustrated.
The embryo material of following examples is hybrid Liriodendron chinense embryonal suspension cell, and its cultural method is with Chinese patent application 02112948.7 or 201010298850.6.The formula of M13 minimal medium is: MS+2, and 4-D 2.0mg/L+6, BA0.2mg/L+LH 500mg/L+sucrose 30g/L, pH value is 5.8.
In order to improve the survival rate of frozen cell, cells,primordial need to be carried out to pretreatment, the measure of taking is that the cells,primordial of the freezing preservation of needs is seeded on the medium that contains the Osmolyte regulator bleeding agents such as sorbierite, make cell partial dehydration by osmotic adjustment, reduce intracellular moisture, the injury that in reduction refrigerating process, the formation of ice crystal causes cell.Detect cell viability by TTC method, thereby determine suitable pretreatment condition.TTC method is the common method that cell viability is measured, and the method is for detection of the activity of dehydrase in cell.Dehydrase makes chlorinated triphenyl four nitrogen (TTC) reduction generate a kind of red complex, and this coloring matter is water insoluble, but is dissolved in alcohol.Therefore, use alcohol extracting, the absorbance while then using spectrophotometric determination 485nm, the cytoactive that absorbance is high is strong, thereby can determine the physiological status of cell.
By embryonal suspension cell subculture in hypertonic liquid medium, the number of days that preculture is different.Hypertonic liquid medium for adding 2.0mg/L 2 in MS minimal medium, 4-D, 0.2mg/L 6, BA, 500mg/L LH and 30g/L sucrose, and concentration is respectively the sorbierite of 0.1mg/L, 0.2mg/L, 0.3mg/L, 0.4mg/L, 0.5mg/L, 0.6mg/L, 0.7mg/L and 0.8mg/L.Result as shown in Figure 1, shows cell survival rate difference under different preculture concentration, the preculture of carrying out during with sorbitol concentration 0.2mol/L～0.5mol/L, and cell survival rate rises and rises gradually with sorbitol concentration, and during to 0.6mol/L, vigor reaches the highest.In the time that sorbitol concentration exceedes 0.6mol/L, cell survival rate is on a declining curve.The highest presentation of results of frozen cell survival rate under 0.6mol/L sorbitol concentration, under this concentration, cell dehydration is the most abundant, does not have height to ooze simultaneously and coerces.
By the comparison of different preculture time, result as shown in Figure 2, show the increase along with the preculture time, vigor after cell freezing obviously rises, under 4d and 7d disposition, cell viability reaches a little peak, and cell viability starts to decline subsequently, and when the preculture time is shorter than 3d, cell viability is obviously on the low side, along with the prolongation cell viability of preculture time constantly raises and tends towards stability, illustrate that embryonal suspension cell oozes a process progressively adapting to of environment to height.When preculture 7d, cell has reached peak under each disposition.Therefore, think that preculture 7d is the suitable time.In suspending and cultivating, under the cell of 1:9 volume ratio and the ratio of medium, cell is the exponential phase in Growth of Cells at 7d, and cell division is vigorous, and morphosis is stable, and to freezing, the damage causing of thawing has stronger resistance.
There is extremely important effect the preculture time to the success of freezing preservation, and by regulating osmotic pressure, the resistance that can improve cell reduces intracellular water content simultaneously, thereby reduces the degree that ice crystal produces, and reduces the injury to cell.By determining take the M13 medium that contains 0.6mol/L sorbierite as pretreatment medium; cell is 1:9 with culture volume ratio; using the M13 minimal medium containing 30% ethylene glycol and 30% glycerine as compound glass protectant; first put into compound glass protectant transition dehydration 0 or the 10min of 60% volume (dilute with water); then putting into 100% compound glass protectant dewatering time 0 ~ 60min is experimental condition, and the preculture time is screened.Result demonstration, at the preculture initial stage, along with the increase of incubation time, cell viability rises rapidly, in the time cultivating 3 days, reaches peak, starts subsequently to decline, and starts cell viability maintain a metastable stage at the 5th day, in the time of 13 days, obviously declines.Along with the increase of preculture time, the vigor after cell freezing obviously rises; At the 4th day and the 7th day, each cell viability of processing reaches a little peak, cell viability starts to decline subsequently, when the preculture time is shorter than 3 days, cell viability is obviously on the low side, along with the prolongation cell viability of preculture time constantly raises and tends towards stability, embody hybridized Chinese tuliptree embryonal suspension cell oozes an environment process progressively adapting to height.Under different transition dehydrations and dewatering time situation, do not passing through the processing of compound glass protectant, directly the cell after preculture is put into liquid nitrogen, along with the increase of preculture time, the resistance of cell strengthens, in rising trend, reached high value at the 8th day, but compared with after treatment through compound glass protectant, its vigor is lower than treated cell, can find at transition dehydration 10min, the dehydration 30min higher cell viability of can living to obtain in each preculture time situation, illustrate to process and can play protective effect to greatest extent to cell.
The freeze box that embodiment 2 is stored to embryonal suspension cell takes out from liquid nitrogen, rapidly cryovial is placed in to the water-bath of different temperatures gradient, and 2min fast thaws.Suck cryoprotector, add the cleaning medium (being pre-culture medium, is the callus proliferated culture medium of 0.6mol/L sorbierite) of 1mL to cryovial, stop 2min, repeat 3 washings.Be provided with five bath temperature gradients at the present embodiment, result as shown in Figure 4, finds that recovery temperature is in the time of 37 ℃, and the vigor of recovery cell peaks.For hybrid Liriodendron chinense, under this temperature condition, the vigor of cell will be higher than the temperature of thawing of 40 ℃.
Promptly return to the environment of normal cultivation from adverse environmental factor by cell by renewal cultivation.Because cryoprotector is that height oozes condition (0.4M sucrose), will cause the too fast expansion of cell if osmotic pressure reduction is too fast, thereby cell membrane produces injury.Therefore reduce lentamente osmotic pressure and can improve to greatest extent cell survival rate, this test procedure process is as follows:
The first step: height oozes renewal cultivation: the cell mass material after washing is transferred to rapidly to the height that is lined with filter paper and ooze recovery media.
A. incubation time gradient be 1,2,3,4,5,6d, 25 ℃ of cultivations.Show according to result of the test, on callus proliferated culture medium, to cultivate 2d comparatively suitable oozing containing the height of 0.4M sucrose.
B. add 0,0.1,0.2,0.3,0.4, the sucrose of 0.5M concentration regulates osmotic pressure.According to result of the test, take sucrose osmotic pressure concentration during as 0.4M, the cell of renewal cultivation can reach rapidly kilter, just can see that new callus grows forwarding to after cultivating 5d in callus proliferated culture medium.
Second step: hypotonic renewal cultivation:
A. cultivated days be 1,2,3,4,5,6d, comparatively suitable containing cultivating 2d on the hypotonic callus proliferated culture medium of 0.3M sucrose.
B. add 0,0.1,0.2,0.3,0.4, the sucrose of 0.5M concentration regulates osmotic pressure, according to test determination when the osmotic pressure concentration 0.3M, the recovery time of cell is the shortest, in callus proliferated culture medium, cultivate 5d and just grow new callus forwarding to, finally transfer on callus proliferated culture medium (being M13 minimal medium) and cultivate.
The somatic embryo of again inducing after recovery, through Cryopreservation and recovery embryo material after treatment, in the upper cultivation of body embryonal induction medium (routine), through the body embryonic development process of 3 months, as shown in Figure 4, can realize high frequency somatic embryo and occur, frozen material growth course is consistent with not having.
Embryonal suspension cell after embodiment 2 is frozen was seeded in respectively the 0.4M sucrose callus proliferated culture medium cultivation that is added with 1mg/L, 2mg/L, 5mg/L, 10mg/L, 20mg/L soluble starch after 2 days, forward on the callus proliferated culture medium of 0.3M sucrose and cultivate 2 days, then forward in normal growth medium.Observed result as shown in Figure 5, finds to be seeded on starch culture-medium effect best, and the retardation of cellular-restoring growth is shorter, is 2～4d, and Growth of Cells is rapid.Other two processing, many cell masses will first become yellowish-brown, just start growth after the retardation of 9～14d.Directly switching is on the medium with soluble starch, and many agglomerates become white or brown, can not grow.
Add the soluble starch of variable concentrations by test, can effectively eliminate moistening phenomenon, cell is damaged in the time carrying out osmotic equilibrium process minimum, result of the test shows that the occurrence frequency of 10mg/L somatic embryo is high.
In the pre-culture medium of embodiment 1, add the ABA of variable concentrations, improve the resistance of cell by ABA, thereby improve the survival rate of cell, in this test, add the ABA of variable concentrations 1mg/L, 2 mg/L, 3mg/L, 4mg/L, 5mg/L in the preculture stage, result as shown in Figure 6, finds can improve significantly the frost resistance of cell in the time of 4mg/L, and cell survival rate has reached 80%.
In tissue, organ and the complete stool experiment of plant, between the accumulation of discovery proline and Osmotic Stress Tolerance, there is significant positive correlation, can be used as and weigh the index of cell to adverse circumstance adaptive capacity.After the sorbierite preculture of variable concentrations, the freeze proof vigor difference to some extent of cell, as shown in Figure 7, after ultralow temperature is preserved, its proline content of cell after 0.1 ~ 0.6mol/L sorbierite is cultivated linearly rises result; Sorbitol concentration 0.5, the proline content at 0.7mol/L place and freezing before almost maintain an equal level, in the time of 0.6mol/L proline content exceed freezing before and reach maximum.Illustrate under condition of ultralow temperature, although cell can Mortality, the proline content in its body still can relative increase be used for Cell protection, this also preserve with ultralow temperature under 0.6mol/L sorbierite preculture after cell viability the highest consistent.
In the pre-culture medium of embodiment 1, add after proline, the survival rate of cell is along with concentration increases and improves.As shown in Figure 8, in the time of 150mg/L, cell survival rate reaches peak, but after proline content continues to increase, cell survival rate starts to decline, illustrate that high concentration proline may produce certain toxic action to cell, cause cell to add outside in high concentration proline situation, cell viability fast-descending.
Claims (8)
1. the superfreeze of an embryo material is preserved method for resuscitation, it is characterized in that, comprises the following steps:
(1) get embryo material to be preserved, by 10% inoculum concentration access hypertonic liquid medium, 25 ℃, cultivate 3～10d; Wherein, hypertonic liquid medium is the M13 minimal medium that is added with 0.1～0.8mg/L sorbierite;
(2) the pre-incubated embryo material of filtration step (1), pack cryovial into, in cryovial, add compound glass protectant, 0 ℃, first with 60% compound glass protectant protection embryo material transition dehydration 0～10min, then forward 100% compound glass protectant dehydration 30min to, then pack the quick-frozen of freeze box direct plunge into Liquid Nitrogen into, the medium-term and long-term preservation of liquid nitrogen; Compound glass protectant is the M13 minimal medium that adds 30% glycerine and 30% ethylene glycol;
(3) from liquid nitrogen, take out freeze box, rapidly cryovial is placed in to 35～43 ℃ of water-baths, 2min fast thaws; Suck compound glass protectant, add the height of 1mL to ooze callus proliferated culture medium to cryovial, stop 2min, for several times repeated washing; It is the M13 minimal medium that adds 0.1～0.5M sucrose that height oozes callus proliferated culture medium;
(4) the embryo material after washing is transferred to rapidly to the height that is lined with filter paper and ooze callus proliferated culture medium, 25 ℃, cultivate 1～6d;
(5) turn embryo material and cultivate 1～6d to hypotonic callus proliferated culture medium, grow new callus, then transfer on callus proliferated culture medium and cultivate; Hypotonic callus proliferated culture medium is the M13 minimal medium that adds 0.1～0.4M sucrose;
(6) shifting length has the embryo material of new callus to cultivate to body embryonal induction medium, until high frequency somatic embryo occurs.
2. preserve method for resuscitation according to the superfreeze of embryo material claimed in claim 1, it is characterized in that: in step (1), cultivate 6～8d.
3. the superfreeze of embryo material according to claim 1 is preserved method for resuscitation, it is characterized in that: in step (1), be also added with 1～5mg/L ABA and 50～200mg/L proline in described hypertonic liquid medium.
4. the superfreeze of embryo material according to claim 1 is preserved method for resuscitation, it is characterized in that: in step (3), and 37 ℃ of water-baths.
5. the superfreeze of embryo material according to claim 1 is preserved method for resuscitation, it is characterized in that: in step (3), it is the M13 minimal medium that adds 0.6mol/L sorbierite that height oozes callus proliferated culture medium.
6. the superfreeze of embryo material according to claim 1 is preserved method for resuscitation, it is characterized in that: in step (4), sucrose concentration is 0.4M, cultivates 2d.
7. the superfreeze of embryo material according to claim 1 is preserved method for resuscitation, it is characterized in that: in step (5), sucrose concentration is 0.3M, cultivates 5d.
8. the superfreeze of embryo material according to claim 1 is preserved method for resuscitation, it is characterized in that: in step (6), cultivate 3 months, just can realize high frequency somatic embryo and occur.
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| CN102823581B (en) * | 2012-09-14 | 2014-08-13 | 上海交通大学 | Screening method of allogenic material for promoting vitrification ultra-low temperature storage |
| CN103283716B (en) * | 2013-06-08 | 2014-11-26 | 中南民族大学 | Simple cryopreservation method of plant BY-2 cell |
| CN103808678A (en) * | 2014-02-25 | 2014-05-21 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | Method and kit for measuring cell activity and application of kit |
| CN103947584B (en) * | 2014-04-04 | 2016-09-28 | 湖南苗王生物科技有限公司 | A kind of defreezing method of Acipenser Sinensis glass frozen essence |
| CN104336009B (en) * | 2014-11-10 | 2016-03-30 | 中国科学院昆明植物研究所 | Poncirus polyandra droplet cryopreservation by vitrification method |
| CN109984123B (en) * | 2019-05-21 | 2021-08-10 | 南京林业大学 | Ultralow temperature preservation method of pine needle brown spot resistant slash pine embryonic callus |
| CN110050784A (en) * | 2019-05-23 | 2019-07-26 | 南京林业大学 | Anti- pine nematode masson pine embryo callus cryopreservation method |
| CN111387176B (en) * | 2020-04-24 | 2021-07-30 | 中国科学院昆明植物研究所 | Vitrification ultralow-temperature preservation method for magnolia officinalis embryonic callus |
| CN111466294B (en) * | 2020-05-29 | 2023-05-30 | 北京林业大学 | Ultralow-temperature preservation method for North China She Songpei sex tissue |
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