CN111758575A - Grape anther embryonic callus differentiation culture medium - Google Patents
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Abstract
The invention provides a grape anther embryonic callus differentiation culture medium, and belongs to the technical field of fruit tree haploid breeding. The medium comprises macroelements such as KNO3、（NH4）2SO4、CaCl2·2H2O、KH2PO4、MgSO4·7H2O; trace elements such as MnSO4·4H2O、ZnSO4·7H2O、H3BO3KI and the like; the iron salt is ferrous glycinate, and the organic components such as inositol, pyridoxine hydrochloride, thiamine hydrochloride, nicotinic acid, biotin, nicotinic acid, riboflavin, glycine, histidine, glutamine and malic acid; other additional components such as Bulbus Lilii cold steep, gamma-aminobutyric acid, L-selenomethionine, phytohemagglutinin, AgNO32-aminopurine, TDZ and allantoin; sucrose and agar are also included. The method obviously improves the differentiation rate of the embryogenic callus of the grape anther, namely the regeneration frequency of embryoid, thereby greatly improving the culture efficiency of the grape anther.
Description
Technical Field
The invention relates to a culture medium for grape anther embryogenic callus differentiation, and belongs to the technical field of fruit tree haploid breeding.
Background
Grape (A)Vitis viniferaL.) is a deciduous vine plant of the genus Vitaceae, and is one of the earliest and most widely-distributed fruit trees cultivated in the world. The grape has large fruit grain, bright color, thick flesh, and sweet and sour taste, and is a fruit with rich nutrition. Grape juice is known as plant milk by scientists. The grape contains protein, saccharide, crude fiber, trace elements such as calcium, phosphorus, ferrum and potassium, vitamins such as vitamin B1, B2, B6 and C, P, and also contains essential components of human bodyMore than ten amino acids and more tartaric acid are needed. The glucose content is up to 10-30%, and glucose is the main component. Therefore, frequent eating of grapes can tonify both neurasthenia and fatigue. The grape contains abundant fruit acid, which is helpful for digestion, and the grape can strengthen spleen and stomach.
The grapes are one of important economic fruit tree crops, have a long cultivation history and rich varieties and are the second largest fruit in the world. China is one of world grape producing countries, and grape cultivation area and yield are in the top of the world. The excellent grape variety is an important guarantee for the continuous development of the grape industry, the breeding level of the grapes is high and low, and the development trend of the grape industry and the strength of market competitiveness are determined by the number of new varieties to be cultivated and the industrialization capability of the excellent grape variety. Because the genetic background of the grape is relatively complex, the traditional crossbreeding mode is long in time consumption and low in efficiency, and has great uncertainty; the asexual propagation seedling raising is carried out by methods such as cuttage or grafting, and the like, and the method is limited by regional and seasonal changes, has a longer period and is easy to accumulate diseases. The development of modern biotechnology such as haploid breeding and the like obviously shortens the breeding period, improves the breeding efficiency and provides a new way for improving grape varieties.
In conventional breeding, 6-7 generations of selfing are generally performed to obtain progeny with stable inheritance. The haploid breeding can obtain a large number of homozygous individuals in a short time, the genetic background is simplified, genetic analysis is facilitated, the target characters are not separated due to generation change, and the extraction and selection efficiency is high. As the gametophyte genotype is rich, the strains from pollen are diversified in plant height, resistance, fertility and the like, and the floral culture strains with different morphological characteristics are formed; in addition, because of the expression of recessive characters of the homozygous material, the selection range of parents is expanded, and breeding resources are enriched. The haploid plant can be obtained by two ways of natural generation and artificial induction. Many fruit trees such as apples, pears, peaches, plums, apricots and the like can naturally generate haploids, but generally, the mutation quantity is small, the frequency is low, and the application in production practice is difficult. In comparison, artificial induction through anther or microspore culture, unpolarized ovary (ovule) culture, distant hybridization and other methods can obviously improve the generation frequency of plant haploid, wherein the anther or microspore isolated culture is most widely applied. Anther culture studies for agricultural use began in 1964 with the anther culture of Datura stramonium from Guha and Maheshwari, followed by a rise in the world. Research on the culture of fruit tree anthers is relatively late, and although great progress has been made in recent years, the progress is still slow compared with crops such as wheat, rice, tobacco, and the like. At present, researches on the flower and drug culture of fruit trees mainly comprise citrus, apples, grapes, litchis, pears, longans, loquats, plums, peaches, jujubes and the like, but the obtained haploid plants are not many.
The development pathways for inducing haploid plants by anther culture in vitro include organogenesis pathways and direct or indirect embryoid body development pathways. The organogenesis approach is a process that under certain conditions, meristematic cells in the callus are differentiated to form buds, then gradually form roots, and finally form complete plants; the embryogenesis pathway refers to the process of differentiation of callus cells to form an embryoid structure containing embryo, radicle and hypocotyl, and finally growth into a complete plant. Compared with the organogenesis approach, the embryoid generation approach has the advantages of high regeneration rate, high regeneration speed and complete structure, and provides a good basis for researching the development, differentiation, totipotency expression, genetic transformation and the like of plant cells.
According to the existing research, the grape anther culture mainly has the following problems: (1) gene dependence, only a few varieties have been reported to obtain haploid plants. (2) The problem of origin of haploid plants remains to be studied further. (3) The yield of the haploid can be greatly improved by regenerating the plant through an embryoid approach, but the induction rate and the differentiation rate of the anther embryogenic callus are lower at present, the generated embryoid is less, and the seedling rate of the embryoid is also lower.
Disclosure of Invention
Aiming at the problems in the prior art, the invention aims to provide a culture medium for differentiating the embryogenic callus of the grape anther, which greatly improves the regeneration frequency of embryoid and further improves the culture efficiency of the grape anther.
The invention is realized by the following technical scheme:
a grape anther embryonic callus differentiation medium is characterized by comprising the following components in use concentration:
macroelements: KNO32150~2350mg/L，（NH4）2SO4233~261mg/L，CaCl2·2H2O 320~350mg/L，KH2PO4150~180mg/L，MgSO4·7H2O 342~370mg/L；
Trace elements: MnSO4·4H2O 15.5~18.3mg/L，ZnSO4·7H2O 3.0~5.0mg/L，H3BO35.8~7.8mg/L，KI 0.8~0.9mg/L，Na2MoO4·2H2O 0.2~0.3mg/L，CuSO4·5H2O 0.02~0.03mg/L，CoCl2·6H2O 0.02~0.03mg/L；
Iron salt: ferrous glycinate 20.5-24.9 mg/L;
organic components: 100-120 mg/L inositol, 4.0-6.0 mg/L pyridoxine hydrochloride, 0.5-1.0 mg/L thiamine hydrochloride, 0.5-1.0 mg/L nicotinic acid, 0.3-0.7 mg/L biotin, 0.3-0.7 mg/L nicotinic acid, 1.0-2.0 mg/L riboflavin, 1.5-2.5 mg/L glycine, 5.0-7.0 mg/L histidine, 80-100 mg/L glutamine, 55-71 mg/L malic acid;
other additional ingredients: 60-80 mL/L of lily leaching liquor, 5-10 mmol/L of gamma-aminobutyric acid, 4.7-6.9 mg/L of L-selenomethionine, 4.5-5.5 mg/L of phytohemagglutinin, and AgNO37.0-9.0 mg/L, 0.6-1.0 g/L of 2-aminopurine, 0.4-0.6 mg/L of TDZ and 1.8-2.2 mg/L of allantoin;
carbon source: 15-25 g/L of sucrose; a coagulant: 5-7 g/L of agar.
The components and the optimal using concentration of the culture medium are as follows:
macroelements: KNO32250mg/L，（NH4）2SO4247mg/L，CaCl2·2H2O 335mg/L，KH2PO4165mg/L，MgSO4·7H2O 356mg/L；
Trace elements: MnSO4·4H2O 16.9mg/L，ZnSO4·7H2O 4.0mg/L，H3BO36.8mg/L，KI 0.85mg/L，Na2MoO4·2H2O 0.25mg/L，CuSO4·5H2O 0.025mg/L，CoCl2·6H2O 0.025mg/L；
Iron salt: ferrous glycinate 22.7 mg/L;
organic components: 110mg/L inositol, 5.0mg/L pyridoxine hydrochloride, 0.75mg/L thiamine hydrochloride, 0.75mg/L nicotinic acid, 0.5mg/L biotin, 0.5mg/L nicotinic acid, 1.5mg/L riboflavin, 2.0mg/L glycine, 6.0mg/L histidine, 90mg/L glutamine, 63mg/L malic acid;
other additional ingredients: 70mL/L of lily leaching liquor, 7.5mmol/L of gamma-aminobutyric acid, 5.3mg/L of L-selenomethionine, 5.0mg/L of phytohemagglutinin and AgNO38.0mg/L, 0.8g/L of 2-aminopurine, 0.5mg/L of TDZ and 2.0mg/L of allantoin;
carbon source: 20g/L of sucrose; a coagulant: 6g/L agar.
The lily leaching liquor is prepared by the following method: weighing 400g of lily bulb, adding 600ml of distilled water, heating and boiling, cooling, filtering with double-layer gauze, and taking the filtrate for later use.
The pH value of the culture medium is adjusted to 5.8-6.0.
Compared with the prior art, the invention has the beneficial effects that:
the culture medium for differentiation of the embryogenic callus of the grape anther is a result of further innovation and improvement based on the existing research and is screened by a large number of tests. The culture medium is optimized in terms of macroelements, microelements and iron salt components and concentration, and is added with multiple organic components such as biotin, nicotinic acid, riboflavin, histidine, glutamine and malic acid, and additional components such as Bulbus Lilii cold extract, gamma-aminobutyric acid, L-selenomethionine, phytohemagglutinin, AgNO32-aminopurine and allantoin, and adopts high-efficiency plant growth regulator TDZ, which has obvious promotion effect on the differentiation of grape embryonic callus into embryoid, and the differentiation rate of the embryonic callus can reach 60.6% at most. The method has positive effects of optimizing a grape anther culture system and accelerating the grape haploid breeding process.
Detailed Description
In order to more fully explain the practice of the present invention, the following preparation method working examples are provided. These examples are merely illustrative and do not limit the scope of the invention.
Example 1
1 materials and methods
1.1 test materials
Embryogenic callus obtained by anther induction culture of 2 grape varieties of Victoria and Imperial concubine rose is used as a test material.
1.2 culture Medium
Differentiation medium: KNO32250mg/L，（NH4）2SO4247mg/L，CaCl2·2H2O 335mg/L，KH2PO4165mg/L，MgSO4·7H2O 356mg/L，MnSO4·4H2O 16.9mg/L，ZnSO4·7H2O 4.0mg/L，H3BO36.8mg/L，KI0.85mg/L，Na2MoO4·2H2O 0.25mg/L，CuSO4·5H2O 0.025mg/L，CoCl2·6H2O0.025 mg/L, ferrous glycinate 22.7mg/L, inositol 110mg/L, pyridoxine hydrochloride 5.0mg/L, thiamine hydrochloride 0.75mg/L, nicotinic acid 0.75mg/L, biotin 0.5mg/L, nicotinic acid 0.5mg/L, riboflavin 1.5mg/L, glycine 2.0mg/L, histidine 6.0mg/L, glutamine 90mg/L, malic acid 63mg/L, Bulbus Lilii leaching solution 70mL/L, gamma-aminobutyric acid 7.5mmol/L, L-selenomethionine 5.3mg/L, AgNO38.0mg/L, TDZ 0.5mg/L, sucrose 20g/L and agar 6 g/L. The pH was adjusted to 5.8.
The lily extract is prepared by the following method: weighing 400g of lily bulb, adding 600ml of distilled water, heating and boiling, cooling, filtering with double-layer gauze, and taking the filtrate for later use.
1.3 culture method
Selecting grape anther whose pollen development period is the mononuclear border period, inoculating, placing grape inflorescence in a refrigerator at 4 deg.C, pretreating at low temperature for 4-5 days, sterilizing, peeling off anther under aseptic condition, inoculating in induction culture medium, and dark culturing in a constant-temperature incubator at 25 deg.C until embryogenic callus is formed. When the embryogenic callus grows to 0.3-0.5 cm, transferring the embryogenic callus to a differentiation medium, culturing at 25 ℃ in the dark for 2 weeks, transferring to the illumination condition (illumination intensity of 2000lx and illumination duration of 16 h/d) for culturing, observing the differentiation condition of the embryogenic callus, counting the number of embryoids, and calculating the differentiation rate. (differentiation rate (%) = number of calli differentiated from embryoid/number of calli inoculated in total × 100).
2 Single factor test
2.1 Effect of phytohemagglutinin on differentiation of embryogenic callus of grape anthers
Phytohemagglutinin was added to the differentiation medium at concentrations of 0, 1.0, 3.0, 5.0, and 7.0mg/L, embryogenic calli obtained from "Victoria" and "Imperial concubine Rose" were transferred and then subjected to differentiation culture, and the differentiation rate was counted for about 2 months, with the results shown in Table 1.
As can be seen from Table 1, the differentiation rate of embryogenic callus showed a tendency of increasing first and then decreasing with the increase of the concentration of phytohemagglutinin. The differentiation rates of the 2 tested materials are respectively 38.9% of Victoria and 46.3% of Imperial concubine rose on a differentiation culture medium added with 5.0mg/L phytohemagglutinin, and are obviously improved compared with a control; on the other hand, the differentiation rate of 2 test materials was close to that of the control on the differentiation medium containing 7.0mg/L of phytohemagglutinin. As can be seen, the optimal concentration of phytohemagglutinin to be added to the differentiation medium was 5.0 mg/L.
2.2 Effect of allantoin on differentiation of grape anther embryogenic callus
Allantoin was added to the differentiation medium at concentrations of 0, 1.0, 2.0, 3.0, and 4.0mg/L, embryogenic calli obtained from "Victoria" and "Imperial concubine Rose" were transferred and then subjected to differentiation culture, and the differentiation rates were counted for about 2 months, with the results shown in Table 2.
As can be seen from Table 2, the differentiation rate of the embryogenic callus of the grape anther was improved to some extent in each differentiation medium to which allantoin was added at different concentrations. The differentiation rates of the 2 test materials were the highest on the differentiation medium supplemented with 2.0mg/L allantoin, namely 39.5% Victoria and 48.3% Imperial concubine rose, and were significantly different from the control. As can be seen, the optimal concentration of allantoin added to the differentiation medium was 2.0 mg/L.
2.Effect of 32-aminopurine on differentiation of embryogenic callus of grape anther
2-aminopurine was added to the differentiation medium at concentrations of 0, 0.4, 0.8, 1.2 and 1.6g/L, embryogenic calli obtained from "Victoria" and "Imperial concubine Rose" were transferred and then subjected to differentiation culture, and the differentiation rate was counted for about 2 months, and the results are shown in Table 3.
As can be seen from Table 3, the embryogenic callus differentiation rate showed a tendency of increasing first and then decreasing with the increase of 2-aminopurine concentration. The differentiation rates of the 2 test materials are the highest on a differentiation medium added with 0.8 g/L2-aminopurine, namely 40.7 percent of Victoria and 52.1 percent of Imperial concubine rose, and are obviously improved compared with a control; on the other hand, the differentiation rate of 2 test materials was lower than that of the control in the differentiation medium containing 1.6g/L of 2-aminopurine. As can be seen, since 2-aminopurine at a high concentration exerts an inhibitory effect on the differentiation of embryogenic callus, the optimal concentration of 2-aminopurine added to the differentiation medium was 0.8 g/L.
Example 2
Effect of different differentiation media on differentiation of embryogenic callus of grape anther
The culture medium of the invention: macroelements: KNO32250mg/L，（NH4）2SO4247mg/L，CaCl2·2H2O 335mg/L，KH2PO4165mg/L，MgSO4·7H2O 356mg/L；
Trace elements: MnSO4·4H2O 16.9mg/L，ZnSO4·7H2O 4.0mg/L，H3BO36.8mg/L，KI 0.85mg/L，Na2MoO4·2H2O 0.25mg/L，CuSO4·5H2O 0.025mg/L，CoCl2·6H2O 0.025mg/L；
Iron salt: ferrous glycinate 22.7 mg/L;
organic components: 110mg/L inositol, 5.0mg/L pyridoxine hydrochloride, 0.75mg/L thiamine hydrochloride, 0.75mg/L nicotinic acid, 0.5mg/L biotin, 0.5mg/L nicotinic acid, 1.5mg/L riboflavin, 2.0mg/L glycine, 6.0mg/L histidine, 90mg/L glutamine, 63mg/L malic acid;
other additional ingredients: 70mL/L of lily cold-soaking liquid, 7.5mmol/L of gamma-aminobutyric acid, 5.3mg/L of L-selenomethionine, 5.0mg/L of phytohemagglutinin and AgNO38.0mg/L, 0.8g/L of 2-aminopurine, 0.5mg/L of TDZ and 2.0mg/L of allantoin;
carbon source: 20g/L of sucrose; a coagulant: 6g/L agar.
Control medium 1: improvement B5+ BA 4.0mg/L + NAA 0.2mg/L + sucrose 2% + agar 0.6% (Zhongchangjie et al "Induction of grape pollen plants").
Control medium 2: MS + BA 0.5mg/L + sucrose 2% + agar 0.6% (Zhouchongjie et al, grape pollen plant Induction).
Control medium 3: b is5+6-BA 1.0mg/L + hydrolyzed casein 500mg/L (Fan Shi Qiang et al "Induction and maintenance of embryogenic callus of grape anther").
Embryonic calli obtained from Victoria and Imperial concubine rose were transferred and cultured by differentiation according to the method of example 1, and the development of embryos was observed, and the differentiation rate of the embryonic calli was counted for about 2 months, and the results are shown in Table 4.
As can be seen from Table 3, the differentiation medium was applied to the callus of grape antherThe influence of differentiation is obvious, when the differentiation culture medium is adopted, the differentiation rate of Victoria is 47.5%, the differentiation rate of Imperial concubine rose is 60.6%, and the differentiation rate is obviously improved compared with 3 control culture media. Control media 1-3 are all in B5The medium of the invention is optimized in the components and concentrations of macroelements, microelements and iron salts, and is also added with a plurality of organic components such as biotin, nicotinic acid, riboflavin, histidine, glutamine and malic acid, and additional components such as lily cold-steep liquor, gamma-aminobutyric acid, L-selenomethionine, phytohemagglutinin and AgNO32-aminopurine, TDZ and allantoin have remarkable promoting effect on the differentiation of the embryogenic calluses of the grapes into embryoids.
Finally, it is noted that the above-mentioned preferred embodiments illustrate rather than limit the invention, and that, although the invention has been described in detail with reference to the above-mentioned preferred embodiments, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the scope of the invention as defined by the appended claims.
Claims (4)
1. A grape anther embryonic callus differentiation medium is characterized by comprising the following components in use concentration:
macroelements: KNO32150~2350mg/L，（NH4）2SO4233~261mg/L，CaCl2·2H2O 320~350mg/L，KH2PO4150~180mg/L，MgSO4·7H2O 342~370mg/L；
Trace elements: MnSO4·4H2O 15.5~18.3mg/L，ZnSO4·7H2O 3.0~5.0mg/L，H3BO35.8~7.8mg/L，KI 0.8~0.9mg/L，Na2MoO4·2H2O 0.2~0.3mg/L，CuSO4·5H2O 0.02~0.03mg/L，CoCl2·6H2O 0.02~0.03mg/L；
Iron salt: ferrous glycinate 20.5-24.9 mg/L;
organic components: 100-120 mg/L inositol, 4.0-6.0 mg/L pyridoxine hydrochloride, 0.5-1.0 mg/L thiamine hydrochloride, 0.5-1.0 mg/L nicotinic acid, 0.3-0.7 mg/L biotin, 0.3-0.7 mg/L nicotinic acid, 1.0-2.0 mg/L riboflavin, 1.5-2.5 mg/L glycine, 5.0-7.0 mg/L histidine, 80-100 mg/L glutamine, 55-71 mg/L malic acid;
other additional ingredients: 60-80 mL/L of lily leaching liquor, 5-10 mmol/L of gamma-aminobutyric acid, 4.7-6.9 mg/L of L-selenomethionine, 4.5-5.5 mg/L of phytohemagglutinin, and AgNO37.0-9.0 mg/L, 0.6-1.0 g/L of 2-aminopurine, 0.4-0.6 mg/L of TDZ and 1.8-2.2 mg/L of allantoin;
carbon source: 15-25 g/L of sucrose; a coagulant: 5-7 g/L of agar.
2. The differentiation medium for embryogenic callus of grape anther according to claim 1, wherein the medium comprises the following components and the optimal concentration for use:
macroelements: KNO32250mg/L，（NH4）2SO4247mg/L，CaCl2·2H2O 335mg/L，KH2PO4165mg/L，MgSO4·7H2O 356mg/L；
Trace elements: MnSO4·4H2O 16.9mg/L，ZnSO4·7H2O 4.0mg/L，H3BO36.8mg/L，KI 0.85mg/L，Na2MoO4·2H2O 0.25mg/L，CuSO4·5H2O 0.025mg/L，CoCl2·6H2O 0.025mg/L；
Iron salt: ferrous glycinate 22.7 mg/L;
organic components: 110mg/L inositol, 5.0mg/L pyridoxine hydrochloride, 0.75mg/L thiamine hydrochloride, 0.75mg/L nicotinic acid, 0.5mg/L biotin, 0.5mg/L nicotinic acid, 1.5mg/L riboflavin, 2.0mg/L glycine, 6.0mg/L histidine, 90mg/L glutamine, 63mg/L malic acid;
other additional ingredients: 70mL/L of lily leaching liquor, 7.5mmol/L of gamma-aminobutyric acid, 5.3mg/L of L-selenomethionine, 5.0mg/L of phytohemagglutinin and AgNO38.0mg/L, 2-aminoPurine 0.8g/L, TDZ 0.5mg/L, allantoin 2.0 mg/L;
carbon source: 20g/L of sucrose; a coagulant: 6g/L agar.
3. The differentiation medium for embryogenic callus of grape anther according to claim 1, wherein said lily extract is prepared by the following method: weighing 400g of lily bulb, adding 600ml of distilled water, heating and boiling, cooling, filtering with double-layer gauze, and taking the filtrate for later use.
4. The differentiation medium for embryogenic callus of grape anther according to claim 1, wherein the pH of said medium is adjusted to 5.8-6.0.
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