CN110384045B - Method for preserving and recovering aseptic material from ice grass leaves - Google Patents
Method for preserving and recovering aseptic material from ice grass leaves Download PDFInfo
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Abstract
The invention discloses a method for preserving and recovering a sterile material from wheatgrass leaves, which comprises the following steps: s1: inoculating the pretreated Bingcao leaves on an induction culture medium, and culturing for 10-15 days to form callus; s2: transferring to subculture medium after callus formation, and respectively refrigerating at room temperature and 4 deg.C or freezing at-20 deg.C or-80 deg.C by slow freezing method for preservation; s3: transferring the callus tissues stored at different temperatures to different resuscitation subculture media respectively for growth, and culturing at normal temperature. The callus preserved at normal temperature, low temperature or ultralow temperature in the method can prolong the preservation time, reduce the subculture, greatly reduce the workload compared with the conventional periodical subculture, and ensure the normal recovery growth of the callus.
Description
Technical Field
The invention relates to the technical field of plant tissue culture and plant resource preservation, in particular to a method for preserving and recovering an wheatgrass leaf-derived sterile material.
Background
Bidens pilosa (Latin article: Agropyron cristatum (L.) Gaertn. (Graminae)) is also known as: the wheatgrass and the wheatgrass belong to perennial dry-grown grasses of the gramineae and the wheatgrass, and the stalks are loose and are suitable for the climates from semi-moist to drought. Naturally occurring wheatgrass rarely forms simple vegetation and is often mixed with other grasses, sedge, non-grasses, and shrubs. It is originated from dry grassland, hillside, hilly land and sand land, and is good forage grass, especially horse and sheep like to eat in green and fresh period, and cattle and camel like to eat, and has high nutritive value, and is a medium fattening feed. The wheatgrass is also rich in natural plant flavonoid compounds, amino acids and the like, and also contains low sodium salt, so that the wheatgrass is beneficial to the health of people. Because of good quality, rich nutrition and good palatability, people and livestock like to eat the food; and because of early green turning, green feed can be provided for grazing livestock earlier. However, due to various factors, the existing natural wheatgrass is gradually reduced, and due to the advantages of high nutritive value, suitability for eating and making feed and the like of the wheatgrass, in recent years, people begin to research the tissue culture of wheatgrass explants so as not to be influenced by environmental conditions and provide excellent culture seedlings for artificial planting at any time.
Plant tissue culture is a technique in which explant materials such as plant organs, tissues, cells or protoplasts are cultured on an artificial culture medium under an aseptic condition, and the explant materials are induced to grow into complete plants under a proper condition. The callus is a key material in plant tissue culture, and can be used for carrying out amplification culture on the callus and differentiating buds or roots to propagate a large number of plant bodies; can also be used as a source of single cells for suspension culture of cells. The conventional callus preservation method is a regular subculture, but the work load is large, and the multiplication of materials is also caused by too many subcultures, so that the search for a proper preservation method has a positive effect on scientific research and teaching, and provides a convenient condition for artificial planting at any time.
Disclosure of Invention
An object of the present invention is to solve at least the above problems and to provide at least the advantages described later.
The invention also aims to provide a method for preserving and recovering the sterile material from the leaves of the wheatgrass, which takes the callus of the wheatgrass leaves as the material, obtains the callus which can be recovered and can normally recover and grow by inducing the callus under different culture mediums and preserving the callus under different temperatures, and the method has long time for preserving the callus and less times of subculture.
To achieve these objects and other advantages in accordance with the purpose of the invention, there is provided a method for prolonging the preservation and resuscitation of a sterile material derived from Bingcao leaves, comprising the steps of:
s1: cutting the pretreated Bingyecao into small segments, inoculating the small segments on an induction culture medium, and culturing for 10-15 days to induce and form callus; the culture conditions are as follows: the temperature is 25-27 ℃, and the illumination is 1200 ℃ and 1500 Lux;
s2: transferring the callus to a subculture medium after the callus is formed, and respectively preserving under the conditions of normal temperature, low-temperature refrigeration at 4 ℃, low-temperature refrigeration at-20 ℃ or ultralow-temperature refrigeration at-80 ℃;
subculture medium stored at different temperatures comprising the following components: MS culture medium 1L, NAA 0.5.5-1 mg, 2,4-D0.3-0.5mg, sucrose 30-35g, mannitol 10-30g, abscisic acid 25-50 μmol, wherein agar 6.5-7.5g is added on the basis of the subculture medium during normal temperature storage and low temperature storage;
s3: transferring the callus tissues stored at different temperatures to different resuscitation subculture media respectively for growth, and culturing at normal temperature.
Preferably, the selecting conditions of the Bingcao leaf in S1 are as follows: selecting young ice grass leaves which are robust in growth and free of diseases and insect pests.
Preferably, the preprocessing in S1 is: cleaning selected wheatgrass leaves, soaking the wheatgrass leaves in 75% alcohol solution for 30-40s, soaking the wheatgrass leaves in 0.1-0.2% mercuric chloride solution for 10-15min, washing the wheatgrass leaves with sterile water for 4-6 times, and placing the wheatgrass leaves on sterile paper to absorb water.
Preferably, the wheatgrass leaves pretreated in the S1 are cut into small sections of 1-1.5 cm; each 50mL tube was inoculated with 2-3 small pieces of Bingcao leaves.
Preferably, the induction medium in S1 comprises the following components: MS culture medium 1L, NAA 0.5.5-1 mg, 2,4-D0.3-0.5mg, sucrose 30-35g and agar 6.5-7.5 g.
Preferably, the callus is cultured by adopting a temperature gradient and illumination gradient method in S1, and the method specifically comprises the following steps: irradiating for 6-8h at 25 deg.C under illumination intensity of 1000Lux every day on day 1-3; irradiating at 27 deg.C for 8-10 hr at illumination intensity of 1100Lux every day on day 4-8 days; on the 9 th to 15 th days, the irradiation is carried out for 10 to 12 hours at the illumination intensity of 1200Lux every day under the condition of 26 ℃.
Preferably, the different resuscitation subcultures in S3 include:
a medium for resuscitation at ambient temperature comprising the following components: MS culture medium 1L, KT 2-2.5mg, NAA 0.5-1mg, 2,4-D0.3-0.5mg, sucrose 25-30g and agar 6.5-7.5 g;
a subculture medium for resuscitation after cryopreservation at 4 ℃ comprising the following components: MS culture medium 1L, NAA 0.5.5-1 mg, 2,4-D0.3-0.5mg, sucrose 25-30g and agar 6.5-7.5 g;
transferring the callus frozen and stored at the low temperature of minus 20 ℃ or the ultralow temperature of minus 80 ℃ to a liquid rejuvenation culture medium, thawing the callus according to a fast melting method, transferring the callus to a solid rejuvenation culture medium for culture, and recovering the growth; the liquid rejuvenation culture medium is prepared by mixing a liquid culture medium and glycerol according to the volume ratio of 0.4-1: 1, the liquid culture medium comprises the following components: MS culture medium 1L, 6-BA0.5-1mg, 2, 4-D1-1.5 mg and cane sugar 25-30 g; the solid rejuvenation medium comprises the following components: MS culture medium 1L, 6-BA0.5-1mg, 2, 4-D1-1.5 mg, sucrose 25-30g and agar 6.5-7.5 g.
Preferably, the fast melting method in S3 specifically includes: and (3) transferring the callus on the liquid rejuvenation culture medium into a refrigerator at 4 ℃ for half a day, then putting the callus into a refrigerator at-20 ℃ for overnight, then putting the callus into a refrigerator at-80 ℃ for long-term storage, taking the callus out when resuscitation is needed, and sequentially putting the callus into water baths at 30 ℃ and 35 ℃ for half an hour respectively to finish thawing.
Preferably, the culture conditions in S3 are: at 25-27 deg.C, light irradiation 1200Lux 1500Lux, 10-12h per day.
The invention at least comprises the following beneficial effects:
the invention takes the callus of the wheatgrass leaves as a source, optimizes the content of each component of the callus through an orthogonal test under different temperature conditions, screens out a culture medium suitable for storing under different temperature conditions, ensures that the formed callus can be normally induced, amplified and differentiated, can still recover the growth of the callus after long-time storage through ultralow temperature storage and recovery, is convenient for scientific research, teaching and artificial planting, provides a test tube material with a normal differentiation function at any time, and provides a sterile material for later research and use.
Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.
Drawings
FIG. 1 shows the growth of the revived calli of Bingcao leaves stored for 2 months at normal temperature;
FIG. 2 is the growth condition of the revived wheatgrass leaf callus preserved for 6 months at low temperature;
FIG. 3 is the growth condition of the revived wheatgrass leaf callus after being frozen for 2 years under ultralow temperature condition;
FIG. 4 is a flow chart of a method of the present invention for extended preservation and recovery of a sterile iceberg leaf-derived material.
Detailed Description
The present invention is described in further detail below to enable those skilled in the art to practice the invention with reference to the description.
It will be understood that terms such as "having," "including," and "comprising," as used herein, do not preclude the presence or addition of one or more other elements or groups thereof.
Example 1
1. Culture medium for prolonging storage life of callus and temperature condition screening
Because plant tissue culture is relatively time consuming and material consuming, preservation of callus is highly desirable in order to save time and the corresponding costs. Different nutrient elements are vital to the growth of plant callus, before the plant callus is preserved, a culture medium MS +0.5mg/L NAA +0.3 mg/L2, 4-D + agar 7g/L is required for preserving the callus formed by the leaves of the wheatgrass, the contents of three substances of sucrose, mannitol and abscisic acid are adjusted on the basis, an orthogonal test is designed into 9 formulas (table 1), and each formula is prepared into 200mL solution. Each test material was repeated twice, each tube containing 20mL of medium. The culture was carried out at normal temperature (25 ℃ C.), cold storage at low temperature (4 ℃ C.) and freezing at ultralow temperature (-80 ℃ C.), after which the growth of each experimental material was observed.
TABLE 1 compositions and corresponding amounts of additional substances in callus preservation formulas designed by orthogonal experiments
(Note: 1% means 10g of addition per 1LMS medium; 2% and 3% and so on)
2. Results
TABLE 2 orthogonal experiments growth of culture media of corresponding formulations at different temperatures
Note: growth is shown as a statistical result of 1 month (30 days) and 2 months (60 days) of inoculation.
As can be seen from Table 2, 3% of sucrose and 1% -3% of mannitol are added on the basis of MS +0.5mg/L NAA +0.3 mg/L2, 4-D + agar 7g/L, and the aim of prolonging the callus of the wheatgrass can be achieved by storing the wheatgrass at normal temperature when ABA is 25/mu mol/L-50 mu mol/L.
The effect of adding different amounts of substances at low temperature (4 ℃) is not great, the growth of the material is inhibited at low temperature, the growth is hardly seen under the freezing condition, and the result is not described in detail because the icing is not favorable for one-to-one observation. The result shows that 3 percent of sucrose, 1 to 3 percent of mannitol and 25 to 50 mu mol/L of ABA can be added into a proper culture medium at normal temperature, and the substances can achieve the purpose of prolonging the preservation of the callus no matter whether the substances are added or not under the condition of cryopreservation. It has been observed that the material can be stored at normal temperature for about 1 year without subculture, and that the refrigerated or frozen material can be stored for as long as 3 to 5 years, or even longer.
Example 2
1. Test materials
Leaf of Bingcao
2. Test method
2.1 preparation of the culture Medium
Preparing MS culture medium mother liquor: preparing mother liquor of macroelements, microelements, iron salts and organic substances according to a conventional MS culture medium formula, greatly enlarging by 10 times, and enlarging the rest by 100 times to prepare corresponding mother liquor, and storing in a refrigerator at 4 ℃.
Preparing auxin mother liquor: dissolving two growth factors, NAA (naphthylacetic acid) and 2,4-D (2, 4-dichlorophenoxyacetic acid), respectively with 1M NaOH or 95% alcohol, diluting with ultrapure water to constant volume to make its concentration be 1mg/mL, and storing the prepared mother liquor in a refrigerator at 4 deg.C.
Preparation of a mother solution of cytokinin: dissolving 6-BA (6-benzylaminopurine) and KT (kinetin) in small amount of 1M HCL, and diluting with ultrapure water to desired volume to obtain 1mg mL-1The prepared mother liquor is stored in a refrigerator at 4 ℃;
preparation of an induction culture medium: MS culture medium +0.5mg/L NAA +0.3 mg/L2, 4-D +30g/L sucrose and 7g/L agar.
Subculture medium stored at different temperatures: MS culture medium +2.0-2.5mg/LKT +0.5-1.0mg/L NAA +0.3-0.5 mg/L2, 4-D +30-35g/L cane sugar, 10-30g/L mannitol, abscisic acid 25-50 μmol/L; wherein, 6.5-7.5g of agar is added on the basis of the subculture medium during normal temperature storage and low temperature storage; agar is not needed to be added when the ultra-low temperature of-20 ℃ or-80 ℃ is preserved.
A medium for resuscitation at ambient temperature comprising the following components: MS culture medium 1L, KT 2-2.5mg, NAA 0.5-1.0mg, 2,4-D0.3-0.5mg, sucrose 30g and agar 6.5-7.5 g;
a subculture medium for resuscitation after cryopreservation at 4 ℃ comprising the following components: MS culture medium 1L, NAA 0.5.5-1 mg, 2,4-D0.3-0.5mg, sucrose 30g and agar 6.5-7.5 g;
a liquid rejuvenation medium for frozen (-20 ℃ C. low temperature or-80 ℃ C. ultra-low temperature) storage of revived callus comprising the following components: MS culture medium 1L, 6-BA0.5-1mg, 2, 4-D1-1.5 mg, sucrose 30 g; a solid rejuvenation medium comprising the following ingredients: MS culture medium 1L, 6-BA0.5-1mg, 2, 4-D1-1.5 mg, sucrose 30g and agar 6.5-7.5 g;
2.2 test methods
A method for prolonging the preservation and recovery of a sterile material derived from the leaves of Bingcao, comprising the steps of:
1) selecting young ice grass leaves which are strong in growth and free of diseases and insect pests, cleaning, soaking in 75% alcohol solution for 30s, then soaking in 0.1% mercuric chloride solution for 10min, then washing with sterile water for 4 times, placing on sterile paper to absorb water and cutting into small sections with the size of 1-1.5cm for direct inoculation, and inoculating 2-3 leaf tissues in a 50mL test tube (the culture medium is: MS culture medium +0.5mg/L NAA +0.3 mg/L2, 4-D +30g/L sucrose +7g/L agar), irradiating for 6h at 25 deg.C under illumination intensity of 1000Lux every day for 1-3 days; irradiating at 26 deg.C for 8h at illumination intensity of 1100Lux every day on day 4-8 days; irradiating for 10h at 27 deg.C and illumination intensity of 1200Lux every day on day 9-10 days to induce callus formation;
2) after callus is formed, transferring the callus to a subculture medium, respectively preserving the callus under the conditions of normal temperature, low temperature of 4 ℃, low temperature of-20 ℃ or ultralow temperature of-80 ℃, and observing the growth condition;
wherein, the subculture medium stored at different temperatures comprises the following components: MS culture medium 1L, NAA 0.5.5-1 mg, 2,4-D0.3-0.5mg, sucrose 30-35g, mannitol 10-30g and abscisic acid 25-50 mu mol L-1And 6.5-7.5g of agar;
3) the callus preserved at various temperatures grows on a recovery subculture medium and is cultured at normal temperature;
a medium for resuscitation at ambient temperature comprising the following components: MS culture medium 1L, KT 2-2.5mg, NAA 0.5-1.0mg, 2,4-D0.3-0.5mg, sucrose 30g and agar 6.5-7.5 g;
a subculture medium for resuscitation after cryopreservation at 4 ℃ comprising the following components: MS culture medium 1L, NAA 0.5.5-1 mg, 2,4-D0.3-0.5mg, sucrose 30g and agar 6.5-7.5 g;
freezing the recovered callus at the low temperature of minus 20 ℃ or the ultralow temperature of minus 80 ℃ to transfer the recovered callus to a liquid rejuvenation culture medium (1L of a liquid culture medium MS culture medium, 0.5-1mg of 6-BA, 1-1.5mg of 2,4-D and 30g of cane sugar), after autoclaving the liquid culture medium, additionally preparing 50% and 80% of glycerol by mass fraction and sterilizing, then proportioning the glycerol and the liquid culture medium according to the proportion of 3: 7, 4: 6 and 5: 5 to obtain a mixed solution of the glycerol and the culture medium, thus obtaining the liquid rejuvenation culture medium, and repeating the formula twice. According to the principle of animal cell recovery, on the basis of slow freezing and fast melting, after the transfer is completed on a super clean bench, the cell is placed in a refrigerator at 4 ℃ for half a day, then placed in a refrigerator at-20 ℃ for overnight, finally placed in a refrigerator at-80 ℃ for overnight, taken out, placed in a water bath kettle at 30 ℃ and 35 ℃ for half an hour in sequence, taken into the super clean bench for liquid culture medium culture for one week, transferred to a solid rejuvenation subculture medium (the solid rejuvenation subculture medium comprises the following components of 1L MS culture medium, 0.5-1mg of 6-BA, 1-1.5mg of 2,4-D, 30g of sucrose and 6.5-7.5g of agar), observed for growth and photographed.
2.3 results
2.3.1 Resuscitation and subculture at Normal temperature
The callus tissues preserved at normal temperature, the types and proportions of hormones were changed, and the growth amounts of 5, 6 and 8, 9 were found to be the greatest after 2 months in 9 subculture media (Table 3) (see Table 4), indicating that the formulation of the subculture media was MS1L + sucrose 30g +2.0-2.5mg/L KT +0.5-1.0mg/L NAA +0.3-0.5 mg/L2, 4-D + agar 7.0g/L, which is the state of the wheatgrass callus tissues cultured at normal temperature for 2 months, as shown in FIG. 1.
TABLE 3 composition and content of different hormones added in callus subculture designed by orthogonal experiment
TABLE 4 orthogonal experiments increase of callus at different temperatures (unit: g) in culture medium of corresponding formulation
2.3.2 Resuscitation and subculture at Low temperature (4 ℃ C.)
After the callus of the wheatgrass stored at low temperature (refrigerated) is transferred to a subculture medium for resuscitation, the observation shows that the callus of the formulas 2 and 3 grows well and the growth amount is the largest (see table 4), which indicates that the formula of the subculture medium is 1L of MS culture medium, 30g of cane sugar, 0.5-1mg/L of NAA, 0.3-0.5mg/L of 2, and 7.0g/L of agar. As shown in FIG. 2, it is the growth status of the wheatgrass callus stored at low temperature for 6 months.
2.3.3 Resuscitation at ultralow temperatures
After the wheatgrass callus preserved at ultralow temperature (freezing) is thawed by water bath and transferred to a liquid culture medium for recovery, the wheatgrass callus is found in the liquid culture medium: the glycerol is 3: 7, browning occurred in either the 50% glycerol or 80% glycerol, and the remaining 4: 6 or 5: 5 were all well grown. Therefore, the addition of 40% or 50% of glycerol to the liquid culture medium for freeze recovery is beneficial to the recovery of the growth of the callus. After the liquid culture medium is cultured for one week, the liquid culture medium is transferred to a solid culture medium containing proper hormone types and concentrations, so that better callus can be obtained, and as can be seen from table 5, the increase amount on formulas 5, 6, 8 and 9 is larger after 2 years of cold transfer after 2 months, which shows that the formula MS culture medium 1L + sucrose 30g +0.5-1mg/L6-BA +1.0-1.5 mg/L2, 4-D + agar 7.0g/L is more suitable for the subculture of the frozen wheatgrass callus. FIG. 3 shows that the callus with good growth after 2 years of ultra-low temperature storage after transfer.
TABLE 5 composition, content and callus increment of different hormones in subculture of frozen callus
Example 3
The wheatgrass callus stored at the low temperature for 6 months in the example 2 and the wheatgrass callus frozen at the low temperature for 2 years are respectively transferred into an illumination incubator (the temperature is 25 ℃, the illumination is 8-10h per day, the culture medium is MS culture medium 1L +0.5mg/L NAA +0.3 mg/L2, 4-D +30g/L sucrose +7.0g/L agar) for culture, and test-tube plantlets are formed in about 45 days.
While the embodiments of the invention have been disclosed above, it is not limited to the applications set forth in the description and the embodiments, which are fully applicable in various fields of endeavor to which the invention pertains, and further modifications may readily be made thereto by those skilled in the art, it being understood that the invention is not limited to the details shown and described herein but is not limited to the particular arrangements shown and described without departing from the general concept defined by the appended claims and their equivalents.
Claims (7)
1. A method for preserving and recovering a sterile material derived from wheatgrass leaves is characterized by comprising the following steps:
s1: cutting pretreated wheatgrass leaves into small segments, inoculating the small segments on an induction culture medium, and culturing for 10-15 days to induce and form callus; the culture conditions are as follows: the temperature is 25-27 ℃, and the illumination is 1200 ℃ and 1500 Lux;
s2: transferring the callus to a subculture medium after the callus is formed, and respectively preserving under the conditions of normal temperature, low-temperature refrigeration at 4 ℃, low temperature refrigeration at-20 ℃ or ultralow temperature refrigeration at-80 ℃;
the subculture medium stored at different temperatures consists of the following components: MS culture medium, KT2.0-2.5mg/L, NAA 0.5.5-1 mg/L, 2,4-D0.3-0.5 mg/L, sucrose 30-35g/L, mannitol 10-30g/L, abscisic acid 25-50 mu mol/L, wherein, when the culture medium is preserved at normal temperature and preserved at low temperature of 4 ℃, agar 6.5-7.5g/L is added on the basis of the subculture medium;
s3: transferring the callus tissues stored at different temperatures to different resuscitation subculture media to grow, and culturing at normal temperature;
an induction medium in S1, consisting of the following components: MS culture medium, NAA 0.5-1mg/L, 2,4-D0.3-0.5 mg/L, sucrose 30-35g/L and agar 6.5-7.5 g/L;
the different resuscitation subcultures in S3 include:
the culture medium for resuscitation subculture at normal temperature comprises the following components: MS culture medium, KT 2-2.5mg/L, NAA 0.5-1mg/L, 2,4-D0.3-0.5 mg/L, sucrose 25-30g/L and agar 6.5-7.5 g/L;
the subculture medium for resuscitation after low-temperature refrigeration preservation at 4 ℃ comprises the following components: MS culture medium, NAA 0.5-1mg/L, 2,4-D0.3-0.5 mg/L, sucrose 25-30g/L and agar 6.5-7.5 g/L;
freezing the callus at-20 deg.C or-80 deg.C, thawing, transferring to liquid rejuvenation culture medium, transferring to solid rejuvenation culture medium, and recovering growth; the liquid rejuvenation culture medium is prepared by mixing a liquid culture medium and glycerol according to the volume ratio of 0.4-1: 1, the liquid culture medium consists of the following components: MS culture medium, 6-BA0.5-1 mg/L, 2, 4-D1-1.5 mg/L and cane sugar 25-30 g/L; the solid rejuvenation culture medium consists of the following components: MS culture medium, 6-BA0.5-1 mg/L, 2, 4-D1-1.5 mg/L, sucrose 25-30g/L and agar 6.5-7.5 g/L.
2. The method for preserving and reviving aseptic material derived from leaves of wheatgrass according to claim 1, wherein the selecting conditions of said wheatgrass leaves in S1 are: selecting young ice grass leaves which are robust in growth and free of diseases and insect pests.
3. The method for preserving and reviving an wheatgrass-derived sterile material according to claim 2, wherein the pretreatment in S1 is: cleaning selected wheatgrass leaves, soaking the wheatgrass leaves in 75% alcohol solution for 30-40s, soaking the wheatgrass leaves in 0.1-0.2% mercuric chloride solution for 10-15min, washing the wheatgrass leaves with sterile water for 4-6 times, and placing the wheatgrass leaves on sterile paper to absorb water.
4. The method for preserving and recovering a sterile material derived from leaves of wheatgrass according to claim 1, wherein the leaves of wheatgrass pretreated in S1 are cut into 1 to 1.5cm pieces; each 50mL tube was inoculated with 2-3 small pieces of Bingcao leaves.
5. The method for preserving and recovering the wheatgrass-derived sterile material according to claim 1, wherein callus is cultured by a temperature gradient and illumination gradient method in S1, and the method comprises the following specific steps: irradiating for 6-8h at 25 deg.C under illumination intensity of 1000Lux every day on day 1-3; irradiating at 27 deg.C for 8-10 hr at illumination intensity of 1100Lux every day on day 4-8 days; on the 9 th to 15 th days, the irradiation is carried out for 10 to 12 hours at the illumination intensity of 1200Lux every day under the condition of 26 ℃.
6. The method for preserving and recovering a sterile material derived from leaves of wheatgrass according to claim 1, wherein the fast-melting method in S3 is specifically: and (3) transferring the callus on the liquid rejuvenation culture medium into a refrigerator at 4 ℃ for half a day, then putting the callus into a refrigerator at-20 ℃ for overnight, then putting the callus into a refrigerator at-80 ℃ for long-term storage, taking the callus out when resuscitation is needed, and sequentially putting the callus into water baths at 30 ℃ and 35 ℃ for half an hour respectively to finish thawing.
7. The method for preserving and recovering a sterile material derived from leaves of wheatgrass according to claim 1, wherein the culture conditions in S3 are: at 25-27 deg.C, light irradiation 1200Lux 1500Lux, 10-12h per day.
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