CN114574422B - Culture medium for inducing wheat anther to heal - Google Patents
Culture medium for inducing wheat anther to heal Download PDFInfo
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- CN114574422B CN114574422B CN202210334792.0A CN202210334792A CN114574422B CN 114574422 B CN114574422 B CN 114574422B CN 202210334792 A CN202210334792 A CN 202210334792A CN 114574422 B CN114574422 B CN 114574422B
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- 239000001963 growth medium Substances 0.000 title claims abstract description 108
- 241000209140 Triticum Species 0.000 title claims abstract description 40
- 235000021307 Triticum Nutrition 0.000 title claims abstract description 39
- 230000001939 inductive effect Effects 0.000 title claims abstract description 19
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims abstract description 120
- 229910052757 nitrogen Inorganic materials 0.000 claims abstract description 59
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims abstract description 43
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims abstract description 31
- 239000008103 glucose Substances 0.000 claims abstract description 31
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- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 29
- 229910052799 carbon Inorganic materials 0.000 claims abstract description 29
- ODKSFYDXXFIFQN-BYPYZUCNSA-N L-arginine Chemical compound OC(=O)[C@@H](N)CCCN=C(N)N ODKSFYDXXFIFQN-BYPYZUCNSA-N 0.000 claims abstract description 25
- 229930064664 L-arginine Natural products 0.000 claims abstract description 25
- 235000014852 L-arginine Nutrition 0.000 claims abstract description 25
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 claims abstract description 25
- 206010020649 Hyperkeratosis Diseases 0.000 claims abstract description 21
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- 238000000034 method Methods 0.000 claims description 15
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- 229960000367 inositol Drugs 0.000 claims description 6
- CDAISMWEOUEBRE-GPIVLXJGSA-N inositol Chemical compound O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](O)[C@@H]1O CDAISMWEOUEBRE-GPIVLXJGSA-N 0.000 claims description 6
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- ZUFQODAHGAHPFQ-UHFFFAOYSA-N pyridoxine hydrochloride Chemical compound Cl.CC1=NC=C(CO)C(CO)=C1O ZUFQODAHGAHPFQ-UHFFFAOYSA-N 0.000 claims description 6
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- DPJRMOMPQZCRJU-UHFFFAOYSA-M thiamine hydrochloride Chemical compound Cl.[Cl-].CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N DPJRMOMPQZCRJU-UHFFFAOYSA-M 0.000 claims description 6
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- 125000000570 L-alpha-aspartyl group Chemical group [H]OC(=O)C([H])([H])[C@]([H])(N([H])[H])C(*)=O 0.000 claims description 2
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/0018—Culture media for cell or tissue culture
- C12N5/0025—Culture media for plant cell or plant tissue culture
Abstract
The invention relates to the technical field of flower cultivation, and provides a culture medium for inducing wheat anther to heal, which is used for adjusting nitrogen sources: removing inorganic reduced nitrogen, and adjusting the reduced nitrogen into organic reduced nitrogen, wherein the organic reduced nitrogen comprises L-glutamine, L-arginine or the organic reduced nitrogen comprises L-glutamine, L-arginine, L-asparagine and glycine; and/or adjusting the carbon source: sucrose was adjusted to glucose. By adjusting the nitrogen source and/or the carbon source, the cure rate of wheat anther is obviously improved. The PCI culture medium designed by the invention takes wheat varieties 'Jinhe 9123' and 'Kenong 199' as test materials, compared with the wheat anther culture medium which is considered to be the best at present, the recovery rate can be improved by at least 20 percent, the recovery time is about one week earlier, the growth speed of the callus is fast, and the superiority of capturing more gene recombinants and improving the efficiency of obtaining haploids when the breeding application is greatly improved is shown.
Description
Technical Field
The invention relates to the technical field of flower cultivation, in particular to a culture medium for inducing wheat anthers to heal.
Background
The anther of the plant is cultivated by a culture medium, the dryness of haploid microspores is mobilized, and the haploid microspores are induced into callus which can be split continuously and has the capability of regenerating complete plants (namely totipotency), so that the method is an important technical key for flower cultivation and breeding of the plant. The aim of this is to eliminate the long filial offspring selfing purification process by doubling the haploid, thus greatly accelerating the breeding process. Since wheat has a complex and large genome, it is objectively decided that wheat cultivars will have more opportunities to retain or select for superior genetic variation and the necessity to accelerate purification by haploid doubling.
Numerous studies and application practices have shown that culture media are the most critical technique for flower cultivation, but the efficiency of flower cultivation is greatly different by using different culture media. As the culture medium for anther culture, there have been reported a few famous culture media such as N6 medium (Zhu to Qing et al, 1975, chinese science, 5:484-490), potato-II medium (Chuang, et al,1978,Proc.Symp.Plant Tissue Cult, sci. Press, peking, p.51-65), C17 medium (Wang Pei et al, 1986, plant journal, 28:38-45), W14 [ Ouyang Junwen et al, 1988, national institute of research, annual report (1987-1988), scientific Press, 102), and the like. In particular to C17 and W14 culture mediums which are specially developed for wheat flower culture. However, in practical flower cultivar applications, the rate of microspore callus induction is still not high enough.
To circumvent the problem of low microspore cure rate that is prominent in anther breeding, some researchers have developed a new approach to the use of distant crosses to cause the loss of male parent chromosomes (Laurie, et al, 1988, theor. Appl. Genet.,76, 393-397) and the use of haploid inducer lines to induce parthenogenetic development [ Ravi, et al, 2010, nature, 464, 615-618; li Liang et al, 2012, university of agriculture, china university, 17 (1): 9-13; zhao, et al, 2013, plant Physiology, 163:721-731; liu et al, 2020, plant Biotech.J., 18:316-318), and the like. While this achieves the desired results and has its remarkable characteristics, induction of haploids by flower culture is still not an alternative, as flower culture does not only obtain haploids, but also has the following unique advantages:
1. genetic recombinants that are not opportunistically present in conventional breeding or other methods of cloning microspores (the number of microspores is large, numerous types are incomparable with other techniques);
2. the new variation which cannot be reserved due to gamete recognition or stigma selection and the like in conventional breeding can be obtained;
3. new variations that are not available by conventional breeding or other methods can be propagated and amplified by culture medium;
4. new variations can be further induced and created at the single cell level by ex vivo culture.
In view of the many irreplaceable advantages of anther culture, anther culture techniques have not been under shaking, and breeders are highly desirous of having a more efficient anther (pollen) medium.
Disclosure of Invention
The invention provides a culture medium for inducing wheat anther to produce a callus, which solves the problem of low rate of producing a callus of a flower cultivated species in the prior art.
The technical scheme of the invention is as follows:
a culture medium for inducing wheat anther to heal,
adjusting a nitrogen source: removing inorganic reduced nitrogen, and adjusting the reduced nitrogen into organic reduced nitrogen, wherein the organic reduced nitrogen comprises L-glutamine, L-arginine or the organic reduced nitrogen comprises L-glutamine, L-arginine, L-asparagine and glycine;
and/or the number of the groups of groups,
and (3) adjusting a carbon source: sucrose was adjusted to glucose.
As a further technical scheme, the nitrogen source comprises the following components in mg/L as mass concentration unit: 500-1200 parts of glutamine, 150-200 parts of L-arginine or 500-1200 parts of glutamine, 150-200 parts of L-arginine, 110-135 parts of L-asparagine and 35-60 parts of glycine;
and/or the number of the groups of groups,
the carbon source comprises the following components: glucose 50000-60000.
The invention also provides a PCI culture medium for inducing the wheat anther to heal, wherein the culture medium is a PCI culture medium, and the formula of the PCI culture medium comprises major elements, nitrogen sources, ferric salts, trace elements, organic components and carbon sources;
the macroelements comprise the following components in mg/L as mass concentration units: KNO (KNO) 3 1300-1700，CaCl 2 ·2H 2 O 120-135，MgSO 4 ·7H 2 O 155-180，KH 2 PO 4 410-500；
The nitrogen source comprises the following components in mg/L (mass concentration unit): 500-1200 parts of L-glutamine, 150-200 parts of L-arginine, 110-135 parts of L-aspartyl and 35-60 parts of glycine;
the carbon source comprises the following components in mg/L (mass concentration unit): glucose 50000-60000.
As a further technical scheme, the formula of the PCI culture medium comprises major elements, nitrogen sources, ferric salts, trace elements, organic components and carbon sources;
the macroelements comprise the following components in mg/L as mass concentration units: KNO (KNO) 3 1500，CaCl 2 ·2H 2 O 130，MgSO 4 ·7H 2 O 160，KH 2 PO 4 450；
The nitrogen source comprises the following components in mg/L (mass concentration unit): l-glutamine 1000, L-arginine 170, L-asparagine 130, glycine 50;
the ferric salt comprises the following components in mg/L (mass concentration unit): feSO 4 ·7H 2 O 27.85，Na 2 -EDTA·2H 2 O 37.25；
The trace elements comprise the following components in mg/L as mass concentration unit: KI 0.415, H 3 BO 3 3.1，MnSO 4 ·4H 2 O 11.15，ZnSO 4 ·7H 2 O 4.3，Na 2 MoO 4 ·2H 2 O 0.125，CuSO 4 ·5H 2 O 0.0125，CoCl 2 ·6H 2 O 0.0125；
The organic components are calculated by taking mg/L as mass concentration units and comprise the following components: inositol 100, biotin 3, nicotinic acid 0.5, pyridoxine hydrochloride 0.4, thiamine hydrochloride 0.5, glycine 2;
the carbon source comprises the following components in mg/L (mass concentration unit): glucose 54000.
The design concept of the PCI culture medium is as follows:
(1) Redesigning macroelement composition
Organic reduced nitrogen (=nh, -NH 2 ) Advantageous effectsEarly and fast growth of callus, but with inorganic reduced Nitrogen (NH) 4+ ) Ammonia formed in coexistence is heavy in toxicity and Ca 2+ While beneficial for protecting cell membranes, it affects microspore cell division; thus, inorganic reduced Nitrogen (NH) 4+ ) Further reduce Ca 2+ The major element composition of the culture medium is redesigned according to the functions and the action properties of other elements, and the specific formula (mg/L) is as follows: KNO (KNO) 3 1500、CaCl 2 ·2H 2 O 130、MgSO 4 ·7H 2 O 160、KH 2 PO 4 450；
(2) Reduced nitrogen using only organic species
Organic reduced nitrogen (=nh, -NH 2 ) The method can induce cells to divide earlier and faster and promote callus to grow faster, so that the reduced nitrogen is completely changed into organic nitrogen, and the reduced nitrogen composition (mg/L) of the culture medium is designed as follows: l-glutamine 1000, L-arginine 170, L-asparagine 130, glycine 50;
(3) Ferric salt using MS culture medium
Iron salts of the media with MS media (mg/L): feSO 4 ·7H 2 O 27.85、Na 2 -EDTA·2H 2 O 37.25；
(4) Microelements using MS culture medium at 1/2 level
The trace elements (mg/L) of the adjustment culture medium are: KI 0.415, H 3 BO 3 3.1、MnSO 4 ·4H 2 O 11.15、ZnSO 4 ·7H 2 O 4.3、Na 2 MoO 4 ·2H 2 O 0.125、CuSO 4 ·5H 2 O 0.0125、CoCl 2 ·6H 2 O 0.0125；
(5) Dedicated organic component using MS medium
The specific organic components of the culture medium are the same as those of the MS culture medium (mg/L): inositol 100, biotin 1.5, nicotinic acid 0.5, pyridoxine hydrochloride 0.4, thiamine hydrochloride 0.5, glycine 2;
(6) Additional biotin
1.5mg/L biotin is added into the culture medium;
(7) Adjusting the type of carbon source in the culture medium
The common sucrose carbon source in the culture medium is changed into glucose carbon source, and the specific dosage is 54000 mg/L;
(8) Additional growth regulator
2, 4-D2 mg/L, KT 0.5.5 mg/L is added in the culture medium, and the dosage of the growth regulator can be adjusted according to actual conditions;
(9) Plant gel as coagulant
The culture medium coagulant is plant gel (Phytagel), and the specific dosage is as follows: 2400mg/L, if the coagulation effect is poor, the dosage can be increased appropriately;
(10) Changing sterilization conditions and modes
After the volume of the culture medium is fixed, the pH value is adjusted to 5.8, and the high-pressure sterilization is carried out under the following sterilization conditions: 115 ℃ for 15 minutes; or (b)
Separately sterilizing glucose and other components of the culture medium, uniformly mixing, and pouring into a dish, namely: dissolving components except glucose in each liter of culture medium in water, fixing the volume to 900mL, and regulating the pH value to 5.8; dissolving glucose in each liter of culture medium into water, fixing the volume to 100mL, and regulating the pH value to 5.8; autoclaving the two parts separately; after sterilization, when the temperature of the two parts is reduced to be close to 50 ℃, uniformly mixing the two parts on an ultra-clean bench, and pouring the mixture into a plate culture medium (the thickness of a plate is 2/3 of the height of a dish bottom); after the flat plate is condensed, the wheat anther can be used for culturing;
(11) Preservation of plate Medium
After the flat culture medium is prepared, the flat culture medium cannot be immediately used, can be stored at normal temperature on the premise of ensuring no pollution, needs to be careful in the preservation process to prevent the culture medium from losing water, and is not suitable to be reused when the culture medium is shrunk to nearly half of the original volume.
The working principle and the beneficial effects of the invention are as follows:
1. in the invention, the inorganic reduced nitrogen is removed by adjusting the nitrogen source of the culture medium, and the reduced nitrogen is adjusted to organic reduced nitrogen (=NH, -NH) 2 ) Can induce cells to divide earlier and faster, promote the growth of callus faster,and/or regulating the carbon source to regulate the sucrose into glucose, so as to obtain a culture medium for inducing the wheat anther to heal, and obviously improving the healing rate of the wheat anther.
2. According to the invention, the PCI culture medium is obtained by optimally designing the formula of the wheat anther culture medium, the wheat variety 'Jinhe 9123' and 'Kenong 199' are used as test materials, compared with the wheat anther culture medium W14 with the best effect currently considered by people, the recovery rate on the PCI culture medium is improved by at least 20 percent (the carbon source of the W14 is changed into glucose, the recovery rate is improved by 10 percent, the recovery rate of the PCI is also improved by at least 10 percent compared with the recovery rate of the W14 with the glucose as the carbon source), and the recovery rate is up to more than 40 percent, so that the recovery rate of the wheat anther is remarkably improved.
3. On PCI culture medium, the anther has early healing time, about one week earlier; the degree of the anther to be healed (namely, the degree of the anther to be healed is determined by the number of the independent microspore callus blocks in a single anther), and the number of the anthers can be tens; the growth rate of the callus is also fast. The PCI culture medium designed by the invention realizes the effect of quick and high yield of microspores in wheat anther culture on wheat anther culture, and shows the superiority of capturing more gene recombinants and improving haploid obtaining efficiency when the breeding application is greatly improved.
Drawings
The invention will be described in further detail with reference to the drawings and the detailed description.
FIG. 1 is a photograph of a representative petri dish of Experimental example 1 of the present invention, taken 5 weeks after inoculation, in which the diameter of the petri dish was 6cm, and a, b, c were the 'Jinhe 9123' anthers, respectivelyC、D、EThe conditions of the culture medium are healed; d. e and f are respectively the anthers of' Kenong 199C、D、EThe growth conditions of the culture medium are recovered;
FIG. 2 is a photograph of a representative petri dish of Experimental example 2 of the present invention, taken 5 weeks after inoculation, of the 'Jinhe 9123' antherIThe growth condition of the culture medium is healed after 6 weeks of inoculation;
FIG. 3 is a photograph of a representative petri dish of Experimental example 2 of the present invention, takenAfter 8 weeks of inoculation, the diameter of the dishes is 6cm, a and b are respectively 'Jinhe 9123' anthers in the figureIE, the condition of healing on the culture medium; c. d is the anther of' Kenong 199I、EThe growth conditions of the culture medium are recovered;
FIG. 4 is a photograph of a representative petri dish of Experimental example 3 of the present invention, taken 8 weeks after inoculation, in which the diameter of the petri dish was 6cm and a and b were 'Jinhe 9123' anthers, respectivelyI、JThe conditions of the culture medium are healed;
FIG. 5 is a photograph (1) of a representative petri dish of Experimental example 4 of the present invention, taken 5 weeks after inoculation, wherein the petri dish has a diameter of 6cm, a, b, c are respectively 'Jinhe 9123' anthers, d, e, f are respectively 'Kenong 199' anthers, andB、C、Dthe growth conditions of the culture medium are recovered;
FIG. 6 is a photograph (2) of a representative petri dish of Experimental example 4 of the present invention, taken 9 weeks after inoculation, wherein the petri dish has a diameter of 6cm and a and b are 'Kenong 199' anthers, inD、GThe growth conditions of the culture medium are recovered;
Detailed Description
The technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by one of ordinary skill in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
A culture medium for inducing wheat anther to heal is prepared by adjusting carbon source, namely adjusting sucrose to glucose with mass concentration of 54000mg/L based on the existing W14 culture medium.
Example 2
On the basis of the existing W14 culture medium, the nitrogen source is adjusted, namely inorganic reduction nitrogen is adjusted to organic reduction nitrogen, and the mass concentration mg/L of each component of the organic reduction nitrogen including L-glutamine and L-arginine is as follows: L-Glutamine 1000, L-arginine 170.
Example 3
On the basis of the existing W14 culture medium, the nitrogen source is adjusted, namely inorganic reduction nitrogen is adjusted to organic reduction nitrogen, the organic reduction nitrogen comprises L-glutamine, L-arginine, L-asparagine and glycine, and the mass concentration mg/L of each component is as follows: l-glutamine 1000, L-arginine 170, L-asparagine 130, glycine 50.
Example 4
On the basis of the existing W14 culture medium, the culture medium for inducing wheat anther to heal is prepared by adjusting a carbon source, namely sucrose into glucose, wherein the mass concentration of the glucose is 54000mg/L, and adjusting a nitrogen source, namely inorganic reduction nitrogen into organic reduction nitrogen, wherein the organic reduction nitrogen comprises L-glutamine, L-arginine, L-asparagine and glycine, and the mass concentration of each component is mg/L: l-glutamine 1000, L-arginine 170, L-asparagine 130, glycine 50.
Example 5
PCI culture medium for inducing wheat anther to heal comprises the following components (mg/L): KNO (KNO) 3 1300，CaCl 2 ·2H 2 O 120，MgSO 4 ·7H 2 O 155，KH 2 PO 4 410, L-glutamine 500, L-arginine 150, L-asparagine 110, glycine 35, feSO 4 ·7H 2 O 27.85，Na 2 -EDTA·2H 2 O 37.25，KI 0.415，H 3 BO 3 3.1，MnSO 4 ·4H 2 O 11.15，ZnSO 4 ·7H 2 O 4.3，Na 2 MoO 4 ·2H 2 O 0.125，CuSO 4 ·5H 2 O 0.0125，CoCl 2 ·6H 2 O0.0125, inositol 100, biotin 3, nicotinic acid 0.5, pyridoxine hydrochloride 0.4, thiamine hydrochloride 0.5, glycine 2, glucose 50000,2,4-D2, KT 0.5, and vegetable gel 2400.
Example 6
PCI culture medium for inducing wheat anther to heal comprises the following components (mg/L): KNO (KNO) 3 1700，CaCl 2 ·2H 2 O 135，MgSO 4 ·7H 2 O 180，KH 2 PO 4 500, L-glutamine 1200, L-arginine 200, L-asparagine 135, glycine 30, feSO 4 ·7H 2 O 27.85，Na 2 -EDTA·2H 2 O 37.25，KI 0.415，H 3 BO 3 3.1，MnSO 4 ·4H 2 O 11.15，ZnSO 4 ·7H 2 O 4.3，Na 2 MoO 4 ·2H 2 O 0.125，CuSO 4 ·5H 2 O 0.0125，CoCl 2 ·6H 2 O0.0125, inositol 100, biotin 3, nicotinic acid 0.5, pyridoxine hydrochloride 0.4, thiamine hydrochloride 0.5, glycine 2, glucose 60000,2,4-D2, KT 0.5, and vegetable gel 2400.
Example 7
PCI culture medium for inducing wheat anther to heal comprises the following components (mg/L): KNO (KNO) 3 1500，CaCl 2 ·2H 2 O 130，MgSO 4 ·7H 2 O 160，KH 2 PO 4 450, L-glutamine 1000, L-arginine 170, L-asparagine 130, glycine 50, feSO 4 ·7H 2 O 27.85，Na 2 -EDTA·2H 2 O 37.25，KI 0.415，H 3 BO 3 3.1，MnSO 4 ·4H 2 O 11.15，ZnSO 4 ·7H 2 O 4.3，Na 2 MoO 4 ·2H 2 O 0.125，CuSO 4 ·5H 2 O 0.0125，CoCl 2 ·6H 2 O0.0125, inositol 100, biotin 3, nicotinic acid 0.5, pyridoxine hydrochloride 0.4, thiamine hydrochloride 0.5, glycine 2, glucose 54000,2,4-D2, KT 0.5, and vegetable gel 2400.
The preparation method of the PCI culture medium comprises the following steps: selecting a 1L clean large beaker, pouring 600-700mL of pure water, sequentially weighing the components of the macroelement and the organic reduction nitrogen, pouring the components into the large beaker to dissolve after the former component is dissolved, and adding CaCl (CaCl) 2 A small amount of pure water (6-10 mL) is needed to be dissolved by using another clean small beaker, and then poured into a large beaker; ferric salt, trace elements, special organic components, biotin and plant growthAdding regulator (prepared into mother liquor and stored at low temperature) into a large beaker according to a certain proportion; dissolving the components except glucose in each liter of culture medium in water, fixing the volume to 900mL in a large beaker, adjusting the pH value to 5.8, and finally weighing the plant gum; glucose per liter of medium was dissolved in pure water using an additional 100mL clean beaker, the volume was set to 100mL, and the pH was adjusted to 5.8 (note using a low ion concentration pH meter probe); pouring the two parts into clean triangular bottles with different specifications according to a plan, sealing with sealing films, and sterilizing at 115 ℃ under high pressure for 15min; after sterilization, when the temperature of the two parts is reduced to be close to 50 ℃, the two parts are evenly mixed on an ultra-clean bench and poured into a flat-plate culture medium (the thickness of the flat plate is 2/3 of the height of the bottom of the dish). After the flat plate is condensed, the wheat anther can be used for culturing. In addition, the basic components of the culture medium can be prepared into powder (become commercial culture medium) according to the process of preparing the culture medium powder, and the powder is dissolved in water and then added with plant growth regulator, glucose and the like for preparation when in use.
Experimental example 1 comparative experiment of culture medium with sucrose as carbon source and glucose as carbon source
1. Materials and methods
The experimental materials are two wheat production varieties of 'Jinhe 9123', 'Kenong 199'. The golden grass 9123 'is not only an excellent variety with high yield, drought resistance and powdery mildew resistance, but also an important breeding parent, a plurality of excellent varieties such as' Malan No. 1 'and the like are bred by using the golden grass 9123' as a female parent, the culture property of the golden grass 9123 'is yet to be determined, if the golden grass 9123' is successful, the variety bred by using the golden grass as a parent is suitable for using biotechnology; 'Kenong 199' is a cultivar with good culturability, which was chosen to ensure success of the comparative experiments.
Healthy intact seeds of the two wheat varieties without diseases and damages are selected and sowed in a greenhouse and a field in a planned way. At the late booting stage (about 1-2d before heading) of wheat plants, cutting off the sheath bud with flag leaf at the lower part of the stem nodule, wrapping with sulfuric acid paper, bagging with plastic bag, and dark preserving in 4-5deg.C refrigerator for 3-7d or more (low temperature pretreatment).
During inoculation, the outside of the sheath bud spike is firstly wiped by 70% alcohol on an ultra-clean bench for disinfection, then leaf sheaths are removed to expose young spikes, and then anthers are pulled by using sterile forceps to be inoculated in a culture medium. The dishes are placed on sterilized paper which has been uniformly drawn with 100 small cells before inoculation, preferably to facilitate identification of the type of black matrix white lines printed. The reference culture mediums are opened simultaneously, anthers from the same young spike are inoculated respectively, and anthers from the same floret are also inoculated respectively as much as possible, so that the consistency of the anthers connected between the culture mediums is ensured.
After anther grafting, the dishes were sealed with parafilm, dark cultured for 3d at 30℃and then transferred to 28℃for further dark culture. During the culture period, the healing condition and the growth condition of the callus are timely checked, and statistics and photographing are carried out.
The medium used: w14 using sucrose as carbon sourceDMarking; w14 using glucose as carbon source was prepared as in example 1EMarking, C17 medium was also used in the experiment, usingCMarking but due toCAndDthe cure rate is relatively low and is equal toDThe differences in (2) are not obvious and are omitted from the "experimental results".
2. Experimental results
After about 4 weeks of incubation, anthers of both test materials were grown onEThe culture medium starts to have "blebs" and then gives rise to a small rice-like callus.
The results of the cure rate of anthers are shown in table 1 and fig. 1:
TABLE 1 recovery from different carbon source media
| Experimental materials | D Medium | E Medium |
| Jinhe (Chinese character) herb9123 | 8% | 20% |
| Kenong 199 | 23% | 33% |
The experiment started in spring 2020 (with autumn broadcast material 2019), repeated 2 batches directly and 3 batches indirectly, and the results were stable.
Experimental example 2 comparative experiment of PCI Medium and W14 Medium with glucose instead of carbon Source
1. Materials and methods
Experimental materials and experimental methods are the same as in experimental example 1;
the medium used: PCI Medium formulation as in example 7, useIMarking; w14 medium with glucose as carbon source was prepared as in example 1EMarking; after the culture medium was prepared, the culture dishes with a diameter of 6cm were each poured with about 15mL of the culture medium.
2. Experimental results
About 3 weeks after inoculation, anthers of both test materials were onIThe culture medium starts to appear like a small bubble; the anther that then goes out of the "blebs" will grow callus of small grain size. The callus can be discharged from one end of the anther body or from two sides of the anther body; the anther which is good is in butterfly shape because a plurality of small callus particles grow out from two sides of the anther; at the position ofEOn the medium, the healing time was about one week later. Under a microscope, the small callus particles obtained by anther are very similar to a white or milky small jade bead.
The results of the cure rate of anthers are shown in Table 2, FIGS. 2-3:
TABLE 2PCI [ (TM)I) W14% of culture medium and glucose as carbon sourceE) Recovery rate on the medium
| Experimental materials | E Medium | I Medium |
| Jinhe 9123 | 20% | 30% |
| Kenong 199 | 29% | 43% |
The experiment starts in summer 2020 (with early spring sowing materials) and is carried out by adding materials growing in a greenhouse in winter, and three batches of experiments are carried out in summer 2021, wherein a plurality of small repeats are arranged in each batch. All experiments show that inIThe anther yield on the culture medium is obviously higher than that of the antherEThe culture medium is at least 10 percent higher.
It has also been found that greenhouse-grown materials may be superior to field-grown materials.
Experimental example 3 comparative experiment of culture medium with organic reduced nitrogen as nitrogen source and organic and inorganic reduced nitrogen as nitrogen source
1. Materials and methods
Experimental materials and experimental methods are the same as in experimental example 1;
the medium used: PCI Medium formulation as in example 4, useIMarking; culture medium with increased inorganic reduced nitrogen (3.2 mmol/L NH over PCI culture medium) 4 + ) By usingJAnd (5) marking.
2. Experimental results
About3. After 4 weeks, anthers of the two test materials were inI、JThe culture medium starts to have "blebs" and then gives rise to a small rice-like callus.
The results of the cure rate of anthers are shown in table 3, fig. 4:
TABLE 3 recovery rates on different Nitrogen Source media
| Experimental materials | I Medium | J Medium |
| Jinhe 9123 | 26% | 10% |
| Kenong 199 | 43% | 20% |
Experimental example 4 Effect of calcium magnesium ion level on wheat anther healing
1. Materials and methods
Experimental materials and experimental methods are the same as in experimental example 1;
the medium used: self-setting culture medium 1BThe formula of the mark is as follows (mg/L): l-glutamine 877, L-arginine 261, L-aspartic acid 266, glycine 73, KCl 2760, NH 4 NO 3 40、Ca（NO 3 ） 2 ·4H 2 O 1044、MgSO 4 ·7H 2 O 810、KH 2 PO 4 410. 1/2MS trace element, MS ferric salt, special organic component, glucose 54000 and vegetable gum 2400, and is characterized by calcium and magnesium ionsHigh concentration of Ca 2+ At a concentration of 4.4mmol/L, mg 2+ The concentration is 3.3mmol/L; c17 medium, use ofCMarking, ca 2+ The concentration is 1.02mmol/L, mg 2+ The concentration is 0.61mmol/L; w14 medium, usedDMarking, ca 2+ The concentration is 0.95mmol/L, mg 2+ The concentration is 0.81mmol/L; self-setting culture medium 2GMarking andBthe culture medium differs only in Ca 2+ The concentration was 0.89mmol/L, mg 2+ The concentration is 0.69mmol/L;
2. experimental results
After 4 weeks of culture, the anthers of the two test materials were investigated, atB、C、DThe callus of the small rice grain type grows on all three culture mediums, but in the following conditionsBThe anther yield on the culture medium is obviously lower thanC、DA culture medium. Furthermore, the callus is inBThe growth rate on the culture medium is also obviously slower than that of the culture mediumC、DCulture medium (see table 4, fig. 5).
TABLE 4 influence of different calcium and magnesium ion concentrations on the healing of wheat anthers (1)
| Experimental materials | B Medium | C Medium | D Medium |
| Jinhe 9123 | 2.5% | 11% | 8.5% |
| Kenong 199 | 3.5% | 11% | 12% |
B、C、DComparative experiments on three media were performed on the 2020 month 4, and the test materials 2019 were sowed in the autumn field and repeated for 2 batches. Two reference varieties, inBEventually only 2% -3% of anthers will heal on the medium, but inC、DThe recovery rate on the culture medium can reach 10 percent. Although the other components of the three media also differ greatly, it is speculated that the primary cause of inhibition of microspore division is due to excessive calcium and magnesium ion concentrations. In particular, calcium, while beneficial in stabilizing cell membranes in existing cognition, high calcium concentrations can affect cell division.
To verifyBThe low recovery rate of the culture medium is caused by high concentration of calcium and magnesium ions, and a supplementary experiment is carried out by using the test materials sowed in the field in early spring 2020DAnd (3) withGThe experiment was repeated for two batches. The result shows that the high concentration of calcium and magnesium is a limiting factor for microspore division, and after the concentration is reduced, the recovery rate is obviously improved, and the effect of the recovery rate is achievedDComparable callus formation levels on the medium and a higher growth rate ratio of callusDThe culture medium was significantly accelerated (see table 5, fig. 6).
TABLE 5 influence of different calcium and magnesium ion concentrations on the healing of wheat anthers (2)
| Experimental materials | D Medium | G Medium |
| Kenong 199 | 10% | 10% |
The foregoing is merely illustrative of the present invention and is not intended to limit the invention, but any modifications, equivalents, improvements or modifications within the spirit and principles of the invention are intended to be included within the scope of the invention.
Claims (6)
1. A culture medium for inducing wheat anther to heal is characterized in that,
adjusting a nitrogen source: removing inorganic reduced nitrogen, and adjusting the reduced nitrogen into organic reduced nitrogen, wherein the organic reduced nitrogen comprises L-glutamine, L-arginine or the organic reduced nitrogen comprises L-glutamine, L-arginine, L-asparagine and glycine.
2. The culture medium for inducing the healing of wheat anthers according to claim 1,
the nitrogen source comprises the following components in mg/L (mass concentration unit): 500-1200 parts of glutamine, 150-200 parts of L-arginine or 500-1200 parts of glutamine, 150-200 parts of L-arginine, 110-135 parts of L-asparagine and 35-60 parts of glycine.
3. The culture medium for inducing the healing of wheat anthers according to claim 1,
the culture medium is a PCI culture medium, and the formula of the PCI culture medium comprises major elements, a nitrogen source, ferric salt, trace elements, organic components and a carbon source;
the macroelements comprise the following components in mg/L as mass concentration units: KNO (KNO) 3 1300-1700，CaCl 2 ·2H 2 O 120-135，MgSO 4 ·7H 2 O 155-180，KH 2 PO 4 410-500；
The nitrogen source comprises the following components in mg/L (mass concentration unit): 500-1200 parts of L-glutamine, 150-200 parts of L-arginine, 110-135 parts of L-aspartyl and 35-60 parts of glycine;
the carbon source comprises the following components in mg/L (mass concentration unit): glucose 50000-60000.
4. A culture medium for inducing wheat anther to heal according to claim 3, wherein the formula of the PCI culture medium comprises major elements, nitrogen sources, ferric salts, trace elements, organic components and carbon sources;
the macroelements comprise the following components in mg/L as mass concentration units: KNO (KNO) 3 1500，CaCl 2 ·2H 2 O 130，MgSO 4 ·7H 2 O 160，KH 2 PO 4 450；
The nitrogen source comprises the following components in mg/L (mass concentration unit): l-glutamine 1000, L-arginine 170, L-asparagine 130, glycine 50;
the ferric salt comprises the following components in mg/L (mass concentration unit): feSO 4 ·7H 2 O 27.85，Na 2 -EDTA·2H 2 O 37.25；
The trace elements comprise the following components in mg/L as mass concentration unit: KI 0.415, H 3 BO 3 3.1，MnSO 4 ·4H 2 O 11.15，ZnSO 4 ·7H 2 O 4.3，Na 2 MoO 4 ·2H 2 O 0.125，CuSO 4 ·5H 2 O 0.0125，CoCl 2 ·6H 2 O 0.0125；
The organic components are calculated by taking mg/L as mass concentration units and comprise the following components: inositol 100, biotin 3, nicotinic acid 0.5, pyridoxine hydrochloride 0.4, thiamine hydrochloride 0.5, glycine 2;
the carbon source comprises the following components in mg/L (mass concentration unit): glucose 54000.
5. A preparation method of a culture medium for inducing wheat anthers to heal is characterized by comprising the steps of,
regulating carbon source, regulating sucrose to glucose, using plant gel as coagulant, separating glucose from other components of culture medium, respectively sterilizing at 115deg.C under high pressure for 15min, and mixing.
6. The method for preparing a culture medium for inducing callus formation of wheat anthers according to claim 5, wherein the carbon source comprises the following components in mg/L mass concentration: glucose 50000-60000.
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