CN111466294B - Ultralow-temperature preservation method for North China She Songpei sex tissue - Google Patents
Ultralow-temperature preservation method for North China She Songpei sex tissue Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- 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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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/40—Afforestation or reforestation
Abstract
The invention discloses a method for ultralow-temperature preservation of North China Larix She Songpei sex tissues, which comprises the step of carrying out liquid gradient pre-culture treatment on North China Larix She Songpei sex tissues (cell line embryogenic callus of North China Larix) with gradually increased osmotic agent concentration. The North China She Songpei sex tissue subjected to gradient pre-culture treatment is subjected to ultralow temperature freezing preservation, and then is subjected to thawing treatment when in use. The method is suitable for long-term ultralow temperature preservation of excellent embryogenic cell lines of larch, and after the method is adopted to preserve She Songpei-sex cells of larch for a long time, the cell survival rate is high, the embryogenic tissue recovery culture is rapid, and the embryogenic callus recovery growth rate is high and reaches more than 66%.
Description
Technical Field
The invention relates to a method for maintaining plant callus, in particular to a method for preserving embryogenic callus of a larch cell line at low temperature, belonging to the technical field of tissue culture of forest tree breeding.
Background
Under normal temperature conditions, long-term subculture of embryogenic cultures often has a number of unavoidable problems: firstly, repeatedly performing secondary preservation for a long time, wherein the secondary cost is high; and, if artificial or environmental disturbances occur during the operation, contamination of the culture is caused, possibly causing significant loss of embryogenic cell lines; more notably, prolonged subculturing may cause somaclonal variation, resulting in a decrease or even a loss of embryogenic capacity of the embryogenic culture. Ultralow temperature cryopreservation technology is a very widely and successful long-term germplasm preservation method currently used, which can effectively suppress the occurrence of mutation, preserve the developmental potential of embryogenic materials, and preserve the integrity of the materials in minimal amounts and at minimal risk. The rapid propagation of somatic embryogenesis adopted abroad has become an important way for large-scale production of conifer high-quality seedlings, and the somatic embryogenesis technology can be combined with a cold preservation method. Plant tissues are stored in liquid nitrogen for a long time, so that long-term clone tests are facilitated.
The existing plant embryogenic callus preservation method mainly comprises a culture subculture preservation method and an ultralow temperature freezing preservation method, wherein the ultralow temperature preservation method needs to pre-culture the tissue before low temperature preservation, so that the callus is properly dehydrated, the water content of cells is reduced, and the division and differentiation of the cells are synchronous, thereby enhancing the stress resistance of the material in the face of severe temperature change and high dehydration in the low temperature preservation process. The common method for ultralow temperature preservation and preculture of conifer embryogenic tissue is to add high-concentration sucrose, sorbitol and the like into a culture medium during preculture to improve osmotic pressure and dehydrate cells so as to achieve the effect of increasing cold resistance.
Although the existing low-temperature freezing preservation method can reduce the genetic variation frequency to a certain extent, ensure the genetic stability and the potential preservation of cell viability and morphogenesis, the embryo tissue recovery and the cell survival rate and the recovery growth rate after thawing are different in different ultralow-temperature freezing preservation methods due to different preculture time, preculture medium components, pretreatment and the like. For example: the invention patent application with the application number of CN201910437126.8 discloses an ultralow temperature preservation method for embryogenic callus of masson pine with pine wood nematode disease resistance, which comprises the steps of placing embryogenic callus of a masson pine cell line in a proliferation culture medium containing sucrose for pretreatment, adding a cryoprotectant into a freezing tube, performing program cooling, taking out the freezing tube after the temperature is reduced to-80 ℃, rapidly adding liquid nitrogen, and performing ultralow temperature preservation; when in use, the embryogenic callus is thawed under the condition that the water bath temperature of the ultralow temperature preserved embryogenic callus is 30-40 ℃, the freezing tube is immediately taken out after ice is completely melted, although the activity of the embryogenic callus cells after thawing at 30-35 ℃ is up to 100%, the appearance and microstructure of the callus after ultralow temperature preserved is not obviously different from that of the callus which normally proliferates, and the callus after low temperature preserved still has the capacity of differentiating to form somatic embryos, but before the freezing preservation, the solid culture is dispersed in liquid and then precultured. The liquid proliferation culture which is preserved in a long-term subculture mode is adopted in the patent, the tissue dispersibility and uniformity are better, the tissue grows in the liquid culture medium for a long time, and the adaptability is stronger.
The invention researches on ultralow-temperature cryopreservation of a North China drop She Songpei cell line, discusses a method suitable for the flow of material pre-culture, cryopreservation modes and the like in ultralow-temperature cryopreservation of North China drop She Songpei culture, and lays a technical foundation for long-term stable and low-preservation of germ plasm resources such as North China drop embryogenic culture.
Disclosure of Invention
The invention aims to provide a method for ultralow temperature preservation of North China She Songpei sex tissue, aiming at the defects of a pretreatment method, a cryoprotectant, a thawing method after ultralow temperature preservation and the like in the ultralow temperature preservation process of the existing plant callus, and the pretreatment culture medium is adjusted to culture so as to improve the preculture efficiency and the recovery growth efficiency. The method is more suitable for cryopreservation of the North China She Songpei callus by liquid gradient pretreatment and adopting the mixed cryoprotectant, the activity of embryogenic callus cells after thawing in a water bath at 37 ℃ is up to 78%, the callus after ultralow temperature preservation has no obvious difference in appearance and microstructure from the callus after normal proliferation, and the callus after low temperature preservation still has the capacity of differentiating to form somatic embryos.
In order to achieve the aim of the invention, the invention provides a method for ultralow-temperature preservation of North China She Songpei sex tissues, which comprises the step of carrying out liquid gradient pre-culture treatment on North China She Songpei sex callus with gradually increased osmotic agent concentration.
Wherein, the liquid gradient pre-culture treatment is to gradually add the penetrating agent into the liquid culture medium in the liquid culture process, and the adding amount of the penetrating agent is increased in a gradient way.
In particular, the osmotic agent is selected from sorbitol or sucrose, preferably sorbitol.
In particular, the concentration of the penetrant in the liquid medium is 0.1 to 0.5mol/L, preferably 0.2 to 0.4mol/L.
In particular, the North China Larix She Songpei callus is selected from the cell line embryogenic callus of North China Larix.
In particular, the North China Larix She Songpei callus is selected from embryogenic suspension callus fluid of a North China Larix cell line.
In particular, the embryogenic suspension callus solution of the larch cell line is prepared and obtained by the following method:
a) Taking immature zygotic embryos of larch as explants, and performing induction treatment on the explants on a solid culture medium to obtain embryogenic callus;
b) Inoculating the embryogenic callus obtained by the induction treatment in the step A) into a liquid multiplication culture medium, carrying out liquid multiplication culture, and carrying out subculture for 1 time every 2 weeks, wherein the subculture mode is that 60-mesh and 100-mesh cell sieves are used for filtering, large cell clusters are filtered, the culture solution is precipitated for 1-2 hours after filtering, the supernatant is discarded, fresh liquid multiplication culture medium is added, and the culture solution obtained by subculture for 3-4 days is the embryogenic suspension callus solution of the larch cell line.
Wherein, the solid culture medium in the step A) is as follows: 1/2LV+2, 4-D1.5-2.5 mg/L+6-BA0.5-1.5 mg/L+30 g/L sucrose+3 g/L vegetable gel+400 mg/L hydrolyzed casein+500 mg/L glutamine, preferably 1/2LV+2,4-D2mg/L+6-BA 1 mg/L+30 g/L sucrose+3 g/L vegetable gel+400 mg/L hydrolyzed casein+500 mg/L glutamine, pH 5.8;
in particular, the culture conditions for the induction culture are: dark culture at 24+ -1deg.C for 6 weeks.
Wherein, the liquid proliferation medium in the step B) is: 1/2LV+2,4-D0.1-0.3mg/L+6-BA 0.05-0.15 mg/L+30 g/L sucrose+400 mg/L casein hydrolysate+500 mg/L glutamine, pH 5.8; preferably 1/2LV+2,4-D0.2mg/L+6-BA 0.1 mg/L+30 g/L sucrose+400 mg/L casein hydrolysate+500 mg/L glutamine.
In particular, the culture conditions of the liquid multiplication culture: dark culture at 24+ -1deg.C.
In another aspect, the invention provides a method for preserving North China She Songpei sex tissue at ultralow temperature, which comprises the following steps in sequence:
1) Placing the North China drop She Songpei callus in a liquid proliferation culture medium containing a penetrating agent for liquid gradient pre-culture treatment;
2) Placing embryogenic callus subjected to liquid gradient pre-culture treatment in a freezing tube, adding a composite cryoprotectant, performing program cooling, taking out after the temperature is reduced to-40 ℃, and then putting into liquid nitrogen for ultralow-temperature preservation;
3) When in use, the freezing tube for storing embryogenic callus at ultralow temperature is taken out for thawing treatment.
Wherein, the liquid gradient pre-culture treatment time in the step 1) is 20-72h, preferably 24-48h, and more preferably 48h.
In particular, the osmotic agent is selected from sorbitol or sucrose, preferably sorbitol.
In particular, the concentration of the penetrant is 0.1 to 0.5mol/L, preferably 0.2 to 0.4mol/L.
Wherein, the North China Larix She Songpei callus in the step 1) is selected from embryogenic callus of a North China Larix cell line.
In particular, the embryogenic callus of the larch cell line is selected from embryogenic suspension callus liquid of the larch cell line.
Wherein, the liquid proliferation culture medium in the step 1) is as follows: 1/2LV+2,4-D0.1-0.3mg/L+6-BA 0.05-0.15 mg/L+30 g/L sucrose+400 mg/L hydrolyzed casein+500 mg/L glutamine, preferably 1/2LV+2,4-D0.2mg/L+6-BA 0.1 mg/L+30 g/L sucrose+400 mg/L hydrolyzed casein+500 mg/L glutamine.
In particular, the liquid gradient pre-culture treatment in step 1) comprises the following steps:
1-1) placing embryogenic callus of a larch cell line in a first preculture medium, and performing preculture treatment for 20-36h in a first stage;
1-2) after the first stage of preculture is finished, taking out the callus, putting the callus into a second preculture medium, and performing the second stage of preculture treatment for 20-36h.
Wherein, the first preculture medium in the step 1-1) is a liquid proliferation medium containing an osmotic agent with a concentration of 0.1-0.3 mol/L.
In particular, the concentration of the penetrant is preferably 0.2mol/L.
In particular, the first preculture medium is 1/2LV+2,4-D0.1-0.3mg/L+6-BA 0.05-0.15 mg/L+30 g/L+30 mg/L of hydrolyzed casein+500 mg/L of glutamine+0.1-0.3 mol/L sorbitol, preferably 1/2LV+2,4-D0.2mg/L+6-BA 0.1 mg/L+30 g/L of sucrose+400 mg/L of hydrolyzed casein+500 mg/L of glutamine+0.2 mol/L sorbitol.
In particular, the first stage preculture time in step 1-1) is preferably 24 hours; the second stage preculture time in step 1-2) is preferably 24 hours.
Wherein, the second preculture medium in the step 1-2) is a liquid proliferation medium containing an osmotic agent with a concentration of 0.3-0.5 mol/L.
In particular, the concentration of the penetrant is preferably 0.4mol/L.
In particular, the second preculture medium is: 1/2LV+2,4-D0.1-0.3mg/L+6-BA 0.05-0.15 mg/L+30 mg/L of sucrose+400 mg/L of hydrolyzed casein+500 mg/L of glutamine+0.3-0.5 mol/L of sorbitol, preferably 1/2LV+2,4-D0.2mg/L+6-BA 0.1 mg/L+30 g/L of sucrose+400 mg/L of hydrolyzed casein+500 mg/L of glutamine+0.4 mol/L of sorbitol.
In particular, the liquid multiplication medium in steps 1-1), 1-2) is: 1/2LV+2,4-D0.1-0.3mg/L+6-BA 0.05-0.15 mg/L+30 g/L sucrose+400 mg/L hydrolyzed casein+500 mg/L glutamine, preferably 1/2LV+2,4-D0.2mg/L+6-BA 0.1 mg/L+30 g/L sucrose+400 mg/L hydrolyzed casein+500 mg/L glutamine.
In particular, after the first stage of the preculture treatment, the method also comprises the steps of filtering by a 60-mesh and 100-mesh cell sieve, filtering the first preculture medium, placing the first preculture medium into a second preculture medium, and performing the second stage of the preculture treatment
Wherein, the compound cryoprotectant in the step 2) is DMSO and sorbitol or DMSO and sucrose; DMSO and sorbitol are preferred.
In particular, the concentration of DMSO in the composite cryoprotectant is less than or equal to 10%, preferably 5-10%; the concentration of sorbitol is 0.3-0.5mol/L, preferably 0.4mol/L; the concentration of sucrose is 0.3-0.5mol/L, preferably 0.4mol/L.
In particular, the composite cryoprotectant is: 0.4mol/L sorbitol+5-10% DMSO or 0.4mol/L sucrose+5-10% DMSO; preferably 0.4mol/L sorbitol+5-10% DMSO.
In particular, the composite cryoprotectant is: 0.4mol/L sorbitol+5% DMSO;0.4mol/L sorbitol+10% DMSO;0.4mol/L sucrose+5% DMSO or 0.4mol/L sucrose+10% DMSO; preferably 0.4mol/L sorbitol+5% DMSO.
Wherein the ratio of the volume of the composite cryoprotectant to the volume of the culture subjected to the pre-culture treatment is (1-4): 1, preferably 2:1.
In particular, the method also comprises the step of filtering culture solution by adopting a 60-mesh and 100-mesh cell sieve after the liquid gradient preculture treatment to obtain the preculture-treated embryogenic callus culture.
In particular, in the step 2), the program cooling is that the freezing pipe is put into a program cooling box for program cooling, the cooling speed is-1 ℃/min, the freezing pipe is put into a refrigerator at the temperature of-40 ℃ and the program cooling is carried out for at least 4 hours until the temperature is reduced to the temperature of-40 ℃.
Wherein, the thawing treatment in the step 3) is to put the freezing tube for preserving embryogenic callus at the ultra-low temperature at room temperature or at the water bath temperature of 30-50 ℃ until the ice is completely thawed.
In particular, the thawing treatment is preferably performed at a water bath temperature of 37 to 42 ℃, and more preferably at 37 ℃.
Because embryogenic callus has higher water content (> 90%), controlling the tissue water loss rate and water loss is critical to the pre-culture effect. The tissue has high water loss speed and large water loss, and excessive dehydration of the tissue can be caused, so that the cell contraction and deformation are caused, the concentration of solutes in the intracellular and extracellular fluids is increased, the pH value is changed, the intracellular environment is deteriorated, and the dehydration injury is caused; and the water loss speed is too slow, the water loss amount is small, the tissue can be frozen in cells due to the too high water content, and the membrane system is damaged during thawing. Therefore, the concentration of the penetrating agent is reduced from low to high in the preculture, the ultralow temperature tolerance of the tissue can be improved, the water loss rate of the tissue is effectively controlled, the water loss environment is adapted, the cell division and differentiation are synchronous, the free water content in the cell is reduced, and the cold resistance is enhanced. Compared with solid preculture, the preculture of the liquid suspension tissue can enable the tissue to be in full contact with the culture medium, so that the tissue or the cells adapting to the gradient hypertonic environment are more, and the cell survival rate of the cryopreservation can be effectively improved. The method of the invention can effectively control the tissue dehydration rate by pre-culturing the embryogenic callus of the larch cell line in the gradient of gradually increasing the concentration of the penetrating agent, so that the tissue can adapt to the dehydration environment and the cell is prevented from being damaged due to too fast dehydration, thus adopting the mode of 0.2mol/L and 0.4mol/L penetrating agent gradient pre-culture in the study.
Compared with the prior art, the method has the following advantages and benefits:
compared with the pretreatment of a single concentration osmotic agent, the pretreatment method adopts the liquid gradient preculture, and the concentration of the osmotic agent is precultured from low to high, so that the water loss rate of the tissue can be more effectively controlled, the tissue can adapt to the water loss environment, the cell is prevented from being damaged too quickly due to water loss, and the ultralow temperature tolerance of the tissue is enhanced. Compared with solid tissues, the liquid tissue has good cell dispersibility, full freeze protection, contribution to cell survival, higher proliferation and maturation efficiency of a liquid suspension system, and contribution to large-scale breeding and application of somatic embryogenesis.
The embryogenic callus of the cell line of the present invention is a stable liquid suspension tissue that is cultured for a long period of time, rather than dispersing solid callus in liquid tissue at a point of time. Therefore, the callus of the invention is suspension liquid tissue, and has good proliferation coefficient, stable proliferation and somatic embryo induction effects.
The tissue is properly dehydrated by pre-culture before ultralow temperature preservation, the water content in cells is reduced, and the division and differentiation of the cells are synchronous, so that the stress resistance of the material in the face of severe temperature change and high dehydration in the low temperature preservation process is enhanced.
The method is suitable for long-term ultralow-temperature preservation of the larch excellent embryogenic cell line, and after the larch She Songpei sex cells are preserved for a long time by adopting the method, embryogenic tissue recovery culture is rapid, and the larch excellent embryogenic cell line can be recovered from the freezing condition within about 1 week; and the recovery growth rate after freezing preservation is more than 66% and is as high as 66.67-70.00% compared with that of the material without freezing preservation; in addition, along with the extension of the freezing time, the recovery growth rate has no obvious difference, so the method is suitable for the long-term ultralow-temperature preservation of the larch excellent embryogenic cell line.
In the method, the composite cryoprotectant of DMSO and sorbitol or DMSO and sucrose is used, the concentration of DMSO is kept between 5 and 10 percent, the toxic action of high-concentration DMSO on embryogenic cells can be reduced, the embryogenic cells can be dehydrated, the cytoplasm reaches a certain concentration, the freezing resistance is provided, and the survival of the frozen cells is facilitated.
The method of the invention adopts liquid proliferation preculture with gradient increase of osmotic agent concentration to embryogenic callus cells, namely, the embryogenic cells are subjected to gradient preculture, the survival rate of the cells after thawing is obviously improved, the survival rate of the cells is improved to more than 61%, and the survival rate reaches 61.45-74.13%.
The method provided by the invention is used for thawing the ultralow-temperature frozen embryogenic cells, the thawing effect is good, the cell survival rate is as high as more than 59%, and reaches 59-78.22%; the thawing speed caused by the thawing temperature is moderate, so that on one hand, the damage of cell water absorption and plasma wall separation is reduced, and on the other hand, the cell breakage and cell death caused by recrystallization in cells due to low thawing speed are avoided to a great extent, thereby remarkably improving the cell survival rate.
Drawings
FIG. 1 shows the effect of different thawing modes on cell viability;
FIG. 2 is a graph showing the recovery growth of North China drop She Songpei callus after ultralow temperature preservation, wherein 2A is the recovery growth for 0 days; 2B is the recovery growth for 7 days; 2C is the recovery of growth for 14 days; 2D is recovered for 28 days;
FIG. 3 is a graph showing the effect of different liquid nitrogen freezing times on the recovery growth rate of embryogenic tissue.
FIG. 4 Induction of somatic embryo maturation after recovery culture of cryopreserved embryogenic tissue
Detailed Description
The invention will be further described with reference to specific embodiments, and advantages and features of the invention will become apparent from the description. These examples are merely exemplary and do not limit the scope of the invention in any way. It will be understood by those skilled in the art that various changes and substitutions of details and forms of the technical solution of the present invention may be made without departing from the spirit and scope of the present invention, but these changes and substitutions fall within the scope of the present invention.
Example 1 test materials
The test material is selected from stable embryo suspension of North China She Songpei cell line BL13-25a for 3-4 days, and the method is concretely characterized by comprising the following steps:
(1) Induction of North China drop She Songpei callus
The immature zygotic embryo of larch is used as an explant to induce embryogenic callus. The embryogenic callus induction solid medium is: 1/2LV+2, 4-D1.5-2.5 mg/L+6-BA0.5-1.5 mg/L+30 g/L sucrose+3 g/L plant gel+400 mg/L hydrolyzed casein+500 mg/L glutamine, preferably 1/2LV+2,4-D2mg/L+6-BA 1 mg/L+30 g/L sucrose+3 g/L plant gel+400 mg/L hydrolyzed casein+500 mg/L glutamine, and after dark culture at 24+ -1deg.C for 6 weeks, embryogenic callus is induced. (LV medium is KNO: KNO) 3 1900mg/L+NH 4 NO 3 1650mg/L+KH 2 PO 4 340mg/L+CaCl 2 .2H 2 O 22mg/L+MgSO 4 .7H 2 O 1850mg/L+H 3 BO 3 31mg/L+ZnSO 4 .7H 2 O 43mg/L+MnSO 4 .H 2 O 21mg/L+Na 2 MoO 4 .2H 2 O 1.25mg/L+KI 4.15mg/L+CuSO 4 .5H 2 O 0.5mg/L+CoCl 2 0.125mg/L+FeSO 4 .7H 2 O 27.8mg/L+Na 2 EDTA.2H 2 O37.3 mg/L+inositol 100 mg/L+vitamin B 1 0.1 mg/L+niacin 0.5 mg/L+vitamin B 6 0.1mg/L)
(2) Preparation of embryogenic suspensions
30ml of liquid multiplication medium is added into a 100ml triangular flask, and the initial inoculum size is 2% (w/v), namely 0.6g of embryogenic callus is added into the medium; wherein:
the liquid proliferation medium is as follows: 1/2LV+2,4-D0.1-0.3mg/L+6-BA 0.05-0.15 mg/L+30 g/L sucrose+400 mg/L hydrolyzed casein+500 mg/L glutamine, preferably 1/2LV+2,4-D0.2 mg/L+6-BA0.1 mg/L+30 g/L sucrose+400 mg/L hydrolyzed casein+500 mg/L glutamine;
liquid proliferation culture conditions: culturing in dark at pH of 5.8 and 24+ -1deg.C at 105 rpm;
every 2 weeks for 1 time by filtering with 60 mesh and 100 mesh cell sieve, filtering to remove large cell mass, precipitating the culture solution for 1-2 hr, discarding supernatant, and adding fresh liquid proliferation culture medium.
The embryogenic suspension of the subculture for 3-4 days is taken as a test material (namely embryogenic callus which is subjected to ultralow temperature cryopreservation, and embryogenic suspension of the North China She Songpei cell line BL13-25a for 3-4 days) for ultralow temperature cryopreservation.
Example 2
1. Pre-culture treatment
According to the pre-cultivation method commonly used for conifers, the study was performed by adding sucrose or sorbitol at high concentration for gradient cultivation. The preculture medium was subjected to gradient preculture with a concentration of 0.2mol/L and 0.4mol/L, respectively, each concentration being cultivated for 24 hours.
1-1) first stage Pre-culture treatment
Taking embryogenic suspension subjected to subculture for 3-4 days as a test material, adding 30ml of a first preculture medium into a 100ml triangular flask, and performing preculture treatment in a first stage; wherein:
the initial inoculum size was 2% (w/v), i.e., 0.6g embryogenic callus was added to the medium;
the first preculture medium was: 1/2LV+2,4-D0.2mg/L+6-BA 0.1 mg/L+30 g/L sucrose+400 mg/L casein hydrolysate+500 mg/L glutamine+0.2 mol/L sorbitol;
first stage preculture treatment conditions: the rotation speed of the shaking table is 105 revolutions per minute, the pH is 5.8, the culture is performed in dark at the temperature of 24+/-1 ℃ for 24 hours (usually 20-36 hours);
1-2) second stage Pre-culture treatment
After 24 hours (usually 20-36 hours) of the first stage preculture treatment, the culture solution is filtered by a 60-mesh and 100-mesh cell sieve and then placed in a second preculture medium for the second stage preculture treatment, wherein:
the second preculture medium was: 1/2LV+2,4-D0.2mg/L+6-BA 0.1 mg/L+30 g/L sucrose+400 mg/L casein hydrolysate+500 mg/L glutamine+0.4 mol/L sorbitol;
second stage preculture treatment conditions: culturing in dark at pH of 5.8 and 24+ -1deg.C at 105 rpm; the incubation time for the second stage pre-incubation treatment is 24 hours (typically 20-36 hours).
After the embryogenic callus suspension material is subjected to gradient pre-culture treatment with gradually increased osmotic agent concentration, embryogenic callus can be more fully contacted with a pre-culture liquid culture medium, so that the embryogenic callus suspension material is more suitable for gradient hypertonic environment tissues or cells, and the cell survival rate of freezing preservation can be effectively improved; and because embryogenic callus water content is higher, control tissue dehydration speed and water loss are the key that influences preculture and cryopreservation effect, compare the preliminary treatment of single concentration osmotic agent, osmotic agent concentration is precultured from low to high gradient, can control the tissue dehydration rate more effectively, make the tissue gradient desiccate, improve the cytoplasmic concentration gradually, adapt to the desiccation environment, avoid the cell desiccation too fast to receive the damage, consequently this patent adopts the osmotic agent gradient preculture's of concentration 0.2-0.4mol/L mode.
2. Freezing treatment
After the second stage of pre-culture treatment for 24 hours (usually 20-36 hours), filtering by adopting a 60-mesh and 100-mesh cell sieve, filtering out culture solution, subpackaging the culture into 2ml of freezing pipes, filling 0.6ml of filtered culture into each freezing pipe, and then adding cryoprotectant with the addition amount of 1.2ml; the ratio of volume of cryoprotectant to culture is 2:1 (typically (1-4): 1) the composition of cryoprotectant is as follows: sorbitol 0.4mol/L+DMSO 5%;
the freezing tube is put into a program cooling box (Nalgene (TM) Cryo 1 ℃ Freezing Container) for program cooling, the cooling speed is-1 ℃/min, and the freezing tube is placed into a refrigerator at-40 ℃. And after the temperature is reduced by at least 4 hours, taking out the freezing tube until the temperature is reduced to-40 ℃, and rapidly putting the freezing tube into a liquid nitrogen biological container for preservation.
3. Thawing treatment
Taking out the freezing pipes stored in the liquid nitrogen biological container for different time (1, 7, 14 and 28 days), and respectively thawing the freezing pipes in a refrigerator at 4 ℃, a water bath at room temperature, a water bath at 37 ℃, a water bath at 42 ℃ and a water bath at 50 ℃; wherein:
respectively standing the frozen tube at 4 ℃ refrigerator and room temperature (25 ℃ and usually 15-30 ℃) until the callus is completely melted;
respectively placing the frozen pipes after the freezing treatment into water baths for thawing, wherein the temperature of the water baths is controlled to be 37 ℃, 42 ℃ and 50 ℃ respectively until the calli are completely re-melted;
after the tissues are remelted, filtering out cryoprotectant, and washing the remelted callus in each freezing tube with a liquid proliferation culture medium for 3-4 times, wherein the liquid proliferation culture medium is as follows: 1/2LV+2,4-D0.1-0.3mg/L+6-BA 0.05-0.15 mg/L+30 g/L sucrose+400 mg/L hydrolyzed casein+500 mg/L glutamine, preferably 1/2LV+2,4-D0.2mg/L+6-BA 0.1 mg/L+30 g/L sucrose+400 mg/L hydrolyzed casein+500 mg/L glutamine;
after the tissue surface liquid medium was blotted with filter paper, it was ready for use. And thawing the re-thawed embryogenic cultures for TTC detection, and detecting the viability of the embryogenic cells after re-thawing the frozen callus, thereby determining the optimal thawing mode.
Example 3
1. Pre-culture treatment
1-1) first stage Pre-culture treatment
The same as in example 2;
1-2) second stage Pre-culture treatment
The remainder was the same as in example 2;
2. freezing treatment
The procedure of example 2 was repeated except that the cryoprotectant was sorbitol 0.4mol/L+DMSO 10%.
3. Thawing treatment
The same as in example 2.
Example 4
1. Pre-culture treatment
1-1) first stage Pre-culture treatment
Except that the first preculture medium was: the same procedure as in example 2 was repeated except that 1/2LV+2,4-D0.2mg/L+6-BA 0.1 mg/L+30 g/L of sucrose+400 mg/L of hydrolyzed casein+500 mg/L of glutamine+0.2 mol/L of sucrose;
1-2) second stage Pre-culture treatment
Except that the second preculture medium was: the same procedures as in example 2 were repeated except that 1/2LV+2,4-D0.2mg/L+6-BA 0.1 mg/L+30 g/L of sucrose+400 mg/L of hydrolyzed casein+500 mg/L of glutamine+0.4 mol/L of sucrose;
2. freezing treatment
The procedure of example 2 was repeated except that the cryoprotectant was sucrose 0.4mol/L+DMSO 5%.
3. Thawing treatment
The same as in example 2.
Example 5
1. Pre-culture treatment
1-1) first stage Pre-culture treatment
Except that the first preculture medium was: the same procedure as in example 2 was repeated except that 1/2LV+2,4-D0.2mg/L+6-BA 0.1 mg/L+30 g/L of sucrose+400 mg/L of hydrolyzed casein+500 mg/L of glutamine+0.2 mol/L of sucrose;
1-2) second stage Pre-culture treatment
Except that the second preculture medium was: the same procedures as in example 2 were repeated except that 1/2LV+2,4-D0.2mg/L+6-BA 0.1 mg/L+30 g/L of sucrose+400 mg/L of hydrolyzed casein+500 mg/L of glutamine+0.4 mol/L of sucrose;
2. freezing treatment
The procedure of example 2 was repeated except that the cryoprotectant was sucrose 0.4mol/L+DMSO 10%.
3. Thawing treatment
The same as in example 2.
Comparative example 1
The test material of the embryogenic suspension subcultured for 3 to 4 days was filtered using 60-mesh and 100-mesh cell sieves, and the culture after the culture broth was filtered was comparative example 1, i.e., without pretreatment and without freezing treatment.
Comparative example 2
The test materials of embryo suspension of subculture for 3-4 days are filtered by a 60-mesh and 100-mesh cell sieve, the culture solution is filtered, the culture is subpackaged into 2ml of freezing pipes, 0.6ml of the filtered culture is filled in each freezing pipe, then freezing treatment is directly carried out, namely, the freezing pipe is put into a program cooling box (Nalgene (TM) Cryo 1 ℃ C. Freezing Container) for program cooling, the cooling speed is-1 ℃/min (usually-1 ℃/min, preferably-1 ℃/min), and the freezing pipe is placed into a refrigerator at-40 ℃. And after the temperature is reduced by at least 4 hours, taking out the freezing tube until the temperature is reduced to-40 ℃, and rapidly putting the freezing tube into a liquid nitrogen biological container for preservation.
The callus of comparative example 2 was directly subjected to freezing treatment without pretreatment and without addition of a cryoprotectant.
Test example 1 cell viability assay
Cell viability of the frozen tissue was determined using triphenyltetrazolium chloride (TTC) reduction (TTC). TTC, 2,3, 5-triphenyltetrazolium chloride (2, 3, 5-triphenoy 1-2H-Tetrazolium Chloride), can be used to determine dehydrogenase activity. Tetrazolium accepts electrons from the electron transport chain in the mitochondria of the cell to form formazan derivatives, thereby turning the original colorless cell into red. The cell activity is strong, the metabolism is vigorous, the transformation capacity is strong, and the color is deep; however, the dead cells are not metabolized to convert tetrazolium into formazan derivatives, and the cells are not discolored. The cells treated with TTC were washed with distilled water to remove residual TTC, and then ethanol was decolorized to elute formazan derivative (red) from the cells to ethanol, and the cells turned colorless. The supernatant was then taken and absorbance was measured. The larger the absorbance, the stronger the viability of the cells. The specific operation is as follows:
thawing and re-thawing the culture in the freezing tube of the ultralow temperature freezing preservation for 1 day in the examples 2-5, respectively placing the washed embryogenic material, the non-frozen callus material of the control example 1 and the frozen and re-thawed callus material of the control example 2 into 10mL test tubes, adding 5mL of 0.5% TTC solution, and culturing for 24 hours under the dark condition at 25 ℃; reducing TTC into water insoluble triphenylmethyl hydrazone (TTF) by dehydrogenase, sucking unreduced TTC solution, and washing 3-4 times with distilled water; adding 5mL of 95% ethanol, and culturing in a constant-temperature water bath at 65 ℃ for 30min; extracting red TTF generated by reducing TTC by dehydrogenase; after cooling, the supernatant was taken and its absorbance at 485nm was measured by a Shimadzu UV2550 spectrophotometer and each treatment was repeated 3 times. The measurement results are shown in Table 1.
Wherein the callus of comparative example 1, which was not subjected to cryopreservation, was used as a control, and the absorbance value measured by the tissue TTC method of comparative example 1 was 100%, and the relative cell viability was calculated according to the formula (1):
the measurement results are shown in Table 1.
TABLE 1 determination of cell viability after callus freezing
From the measurement results, it was found that:
1. the method for ultralow temperature preservation of plant callus has high survival rate of tissue cells after freezing and re-thawing, which reaches more than 61.45%, and the survival rate of the plant callus cells preserved by the method of example 2 reaches 74.13%.
2. Compared with control 2, i.e. the callus of the control group is not pre-treated and no cryoprotectant is added during freezing, the survival rate of the tissue cells directly subjected to freezing preservation is significantly lower than that of the tissue cells treated by the method of the invention, and the average cell survival rate is 23.54% (the ratio is that the tissue directly placed in liquid nitrogen without any treatment is compared with the tissue not subjected to liquid nitrogen preservation). Freezing at low temperature can freeze water in cells, causing irreversible damage to cell structures. The plant tissue is not pretreated, which leads to cell death and thus lower cell viability.
To further investigate the effect of the pre-incubation mode and cryoprotectant type, a two-factor analysis of variance was performed, the results are shown in table 2. From the analysis result, the concentration of the cryoprotectant has a very significant effect on the cell viability (P < 0.01), and since the cryoprotectant DMSO used in the experiment has a certain toxic effect on cells, the high concentration of DMSO can cause cell death, so that 5% DMSO is more suitable for the cryoprotection of the She Songpei calli of North China; the type of osmotic agent, including sorbitol or sucrose, has a significant effect on cell viability (P < 0.05).
TABLE 2 analysis of variance table of pretreatment factors before ultra-low temperature freezing
From the two-factor analysis of variance results of table 2, it can be seen that: the preservation method of the embodiment 2 of the invention, namely 0.2mol/L and 0.4mol/L sorbitol are respectively pre-cultured for 24 hours, and 5% DMSO is added to 0.4mol/L sorbitol as a cryoprotectant, so that the preservation method is more beneficial to the survival of cells after the North China drop She Songpei callus is frozen.
Test example 2 Effect of thawing means on cell viability test
In the ultralow temperature preservation process of plant materials, freezing and thawing can possibly cause freezing injury of cells, and a proper thawing mode is adopted to avoid damage to a cell membrane system caused by severe osmotic pressure change due to moisture absorption in the thawing process.
Thawing test is carried out on plant materials which are subjected to ultralow temperature cryopreservation for 1 day in the embodiment 2, and the freezing pipes which are frozen for 1 day are respectively placed in a refrigerator at 4 ℃ and at room temperature; placing in a water bath kettle at 37deg.C and 42deg.C and 50deg.C, thawing in water bath, thawing in the above five ways, filtering out cryoprotectant after tissue is thawed again, and washing the tissue in each freezing tube with liquid proliferation culture medium for 3-4 times. After the filter paper absorbs the liquid culture medium on the surface of the tissue, the filter paper is used for TTC detection, so that the optimal thawing mode is determined, and the measurement result is shown in figure 1.
As can be seen from fig. 1: along with the gradual rise of the thawing temperature, the cell survival rate shows the trend of rising and then reducing, the thawing effect is 37 ℃ water bath >42 ℃ water bath > room temperature >50 ℃ water bath >4 ℃ refrigerator, wherein the thawing effect is best with 37 ℃ water bath, the cell survival rate is 78.22% on average, and the thawing speed possibly caused by the temperature is moderate, so that the damage of water absorption and separation of the plasma wall of the cells is reduced, and the cell rupture and cell death caused by recrystallization in the cells due to slow thawing speed are avoided to a great extent. Thawing in a refrigerator at 4℃is too slow, causing recrystallization in the cells and thus lower cell viability.
Test example 3 detection of tissue recovery growth after freezing
The most efficient ultra-low temperature cryopreservation procedure obtained from the above experiments was: adding 0.2mol/L and 0.4mol/L sorbitol into the culture medium, and pre-culturing for 24h respectively; taking 0.4mol/L sorbitol and 5% DMSO as cryoprotectants, and performing liquid nitrogen cryopreservation after programmed cooling; after 1 week, frozen embryogenic tissue was removed and thawed in a 37 ℃ water bath; the liquid medium was washed 3 times.
Thawing test was performed on the plant material of example 2 which was stored at ultra-low temperature for 7 days, and the frozen tube after 7 days was placed in a 37℃water bath, thawed in a water bath, and rinsed 3 times with liquid medium.
After the above procedure, the surface of the thawed embryogenic tissue is smoother and has no filiform structure (as shown in fig. 2A), and the tissue is placed in a solid multiplication medium: 1/2LV+2,4-D0.1-0.3mg/L+6-BA 0.05-0.15 mg/L+30 g/L of sucrose+400 mg/L of hydrolyzed casein+500 mg/L of glutamine+2.8 g/L of plant gel, preferably 1/2LV+2,4-D0.2mg/L+6-BA 0.1 mg/L+30 g/L of sucrose+400 mg/L of hydrolyzed casein+500 mg/L of glutamine+2.8 g/L of plant gel, and performing recovery culture at 25deg.C in dark condition; after 7 days of recovery culture, a white translucent filamentous embryogenic callus was first grown on a portion of the embryogenic tissue (see FIG. 2B); after 14 days of recovery culture, embryogenic callus grew out and increased continuously in each part of frozen callus (see FIG. 2C); by the time of recovery culture for 28 days, a layer of regrown embryogenic callus has been packed on the surface of the frozen embryogenic tissue (see FIG. 2D).
Test example 4 test of Effect of cryopreservation time on tissue recovery growth Rate
The aim of ultralow temperature preservation is to preserve excellent germplasm safely and stably for a long time. Therefore, the effect of the ultralow temperature storage time on the recovery growth of the thawed tissue was investigated in this experiment.
Thawing test was performed on plant materials obtained by cryopreservation at ultra-low temperature for 1, 7, 14, and 28 days in example 2, and the frozen tube was placed in a water bath kettle at 37℃and washed 3 times with liquid medium after thawing in water bath.
Transferring the thawed and re-melted embryogenic culture to a solid multiplication medium (the solid multiplication medium is 1/2LV+2,4-D0.2mg/L+6-BA 0.1 mg/L+30 g/L sucrose+400 mg/L casein hydrolysate+500 mg/L glutamine/+2.8 g/L plant gel); 6 embryogenic calli were inoculated per dish, 5 replicates. And (3) performing recovery culture of the embryogenic callus after ultralow temperature freezing under the dark condition at 25 ℃.
In the culture process, the recovery growth condition of the callus is continuously observed, the recovery growth rate is counted after 10 days of culture, the recovery growth rate of the callus is calculated according to a formula (2), and the measurement result is shown in figure 2. Proliferation culture of embryogenic callus without cryopreservation was used as a control.
As can be seen from fig. 3, the growth rate of the embryogenic tissue that was not subjected to the ultralow temperature preservation was 100%; after the embryogenic tissues are frozen and preserved for 1, 7, 14 and 28 days, thawing and restoring culture are carried out, no obvious difference is found between the restoring growth rates after four preservation times, and compared with a control group which is not preserved at ultralow temperature, the restoring growth rate of the embryogenic tissues treated by the method is reduced to 66.67-70.00%. The preservation time of liquid nitrogen has no obvious influence on the recovery growth of the North China She Songpei callus, and the ultralow-temperature preservation condition of the method can achieve the purpose of long-term preservation.
Test example 5 maturation test of somatic embryos
Mature culture of somatic embryos was performed with the tissue subjected to ultralow temperature preservation of example 2, the plant material subjected to ultralow temperature cryopreservation of example 2 for 1, 7, 14, 28 days was thawed and growth culture was resumed, and the callus recovered to growth was placed on a maturation medium for differentiation culture, and the callus differentiation capacity was observed, wherein the tissue recovery culture method was referred to test example 4.
The maturation process of somatic embryos is provided with a transitional culture stage and a maturation culture stage. Uniformly dispersing the liquid suspension tissue on filter paper, filtering out residual liquid culture medium in vacuum, placing on a transition culture medium, and performing dark culture for 7 days at 24+/-1 ℃ under the conditions of 1/2LV culture medium, 2g/L of Activated Carbon (AC) and 10g/L of sucrose without adding plant growth regulator.
In the mature stage, a 1/2LV culture medium is taken as a basic culture medium, and ABA 16mg/L and sucrose 50g/L are added; phytagel7g/L; PEG 75g/L, additional hydrolyzed casein 400mg/L, glutamine 500mg/L, pH 5.8, dark culture at 24+ -1deg.C for about 8 weeks, mature somatic embryo after maturation culture as shown in figure 4.
As can be seen from fig. 4, the embryogenic tissue which is recovered to grow by ultra-low temperature freeze protection can induce somatic embryos with normal structures, and the embryogenic tissue with the somatic embryo regeneration capacity is 100%; in contrast, the surviving tissue directly preserved in liquid nitrogen loses the ability to form normal somatic embryos. Therefore, the cryopreservation treated by the cryoprotection not only maintains the proliferation and growth capacity of the embryogenic tissue, but also does not reduce the differentiation potential of the embryogenic tissue.
The above-described embodiments of the present invention are merely exemplary and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes and substitutions of details and forms of the technical solution of the present invention may be made without departing from the spirit and scope of the present invention, but these changes and substitutions fall within the scope of the present invention.
Claims (5)
1. The method for ultralow-temperature preservation of the North China She Songpei sex tissue is characterized by comprising the following steps in sequence:
1) Subjecting the North China drop She Songpei callus to liquid gradient pre-culture treatment in a liquid proliferation medium containing a penetrating agent, wherein:
the liquid gradient pre-culture treatment is to gradually add the penetrating agent into the liquid culture medium in the liquid culture process, and the adding amount of the penetrating agent is increased in a gradient way, and the method comprises the following steps:
1-1) placing embryogenic callus of a cell line of larch in a first preculture medium, and performing liquid preculture treatment for 20-36h at a first stage, wherein the first preculture medium is a liquid proliferation medium containing an osmotic agent with the concentration of 0.1-0.3 mol/L;
1-2) taking out the callus after the first-stage liquid preculture is finished, and then placing the callus in a second preculture medium for second-stage liquid preculture treatment for 20-36h, wherein the second preculture medium is a liquid proliferation medium containing an osmotic agent with the concentration of 0.3-0.5 mol/L;
the liquid proliferation culture medium is as follows: 1/2LV+2,4-D0.1-0.3mg/L+6-BA 0.05-0.15 mg/L+30 g/L sucrose+400 mg/L hydrolyzed casein+500 mg/L glutamine; the penetrating agent is sorbitol or sucrose;
2) Filtering by adopting a 60-mesh and 100-mesh cell sieve after liquid gradient pre-culture treatment, filtering culture solution, placing the obtained embryogenic callus subjected to the liquid gradient pre-culture treatment into a freezing tube, adding a composite cryoprotectant, performing program cooling, taking out after the temperature is reduced to-40 ℃, and then putting into liquid nitrogen for ultralow temperature preservation, wherein the composite cryoprotectant is as follows: DMSO and sorbitol or DMSO and sucrose, wherein the concentration of DMSO in the composite cryoprotectant is less than or equal to 10%; the concentration of the sorbitol is 0.3-0.5 mol/L; the concentration of the sucrose is 0.3-0.5 mol/L;
3) When in use, the freezing tube for storing embryogenic callus at ultralow temperature is taken out for thawing treatment.
2. The method of claim 1, wherein the osmotic agent is sorbitol.
3. The method of claim 1 or 2, wherein the liquid multiplication medium in step 1) is: 1/2LV+2,4-D0.2mg/L+6-BA 0.1 mg/L+30 g/L sucrose+400 mg/L casein hydrolysate+500 mg/L glutamine.
4. The method of claim 1 or 2, wherein the concentration of the osmotic agent in the liquid propagation medium in step 1) is 0.1 to 0.5mol/L.
5. The method of claim 1 or 2, wherein the concentration of the osmotic agent in the liquid propagation medium in step 1) is 0.2 to 0.4mol/L.
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