CN104082146A - Method for inducing polyploid through wild jujube adventitious buds - Google Patents
Method for inducing polyploid through wild jujube adventitious buds Download PDFInfo
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- CN104082146A CN104082146A CN201410334141.7A CN201410334141A CN104082146A CN 104082146 A CN104082146 A CN 104082146A CN 201410334141 A CN201410334141 A CN 201410334141A CN 104082146 A CN104082146 A CN 104082146A
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Abstract
The invention provides a method for inducing polyploid through wild jujube adventitious buds, relates to the biotechnical field, and the problem that homozygous tetraploid is hard to obtain through existing methods is solved. The method provided by the invention comprises the following steps: starting of cultivation, induction culture, elongating cultivation, strong seedling cultivation and detection on young leaves. The method can be used for cultivating wild jujube homozygous polyploid so as to support the genetic improvement of jujube trees.
Description
Technical field
The present invention relates to biological technical field, refer to especially a kind of method of wild jujube indefinite bud approach induction polyploid.
Background technology
Jujube (Zizyphus.jujuba Mill.) is Rhamnaceae (Rhamnaceae) zizyphus (Zizyphus.Mill.) plant, to originate in Chinese characteristic advantage economic tree, its delicious flavour, fruit shape is attractive in appearance, be rich in several amino acids and the vitamin of needed by human body, also containing the medicinal ingredients such as multiple polysaccharide, trace element and cancer-resisting substance cyclic adenosine monophosphate, is medicine-food two-purpose invigorant.
Wild jujube (Ziziphus spinosus Hu.) originates in North China, how wild, drought-resistant, barren, in the exploitation of mountain area, once plays a significant role.The wild jujube florescence is long, is good nectariferous plant, plants that benevolence is full is used as medicine.Wild jujube is as food, and nutritive value is very high, contains the various trace elements such as potassium, sodium, iron, zinc, phosphorus, selenium; The more important thing is and in wild jujube, contain a large amount of vitamin Cs, its content is 2～3 times of common red date, 20～30 times of oranges and tangerines, and in human body, availability can reach 86.3%.In addition, the natural health nourishing such as fruit juice, fruit vinegar food is liked by consumers in general deeply in recent years, and wild jujube is as producing raw material, and the increase of its jujube fruit matter can significantly be enhanced productivity, and reduces production cost.
There are reports in the polyploid research of wild jujube, is the in-vitro inducing approach of clump bud, stem section but adopt field callus approach or material more, and the chimera that these approach can form conventionally, is difficult to obtain the tetraploid of isozygotying.The present invention, using the young leaflet tablet of wild jujube aseptic seedling as test material, adopts Direct Regeneration indefinite bud approach to carry out the induction of polyploid, thereby effectively avoids chimeric appearance.In addition, correlative study for the in vitro multiploid induction optimization process of Chinese Jujube period have not been reported, the present invention adopts two-steps tissue culture method, in the first step pre-(secretly) cultivation, find first colchicin optimization process period, and then can improve efficiency of inducing mutation, to obtaining a large amount of polyploid materials in the short time, the genetic improvement of jujube tree is had to important value.
Summary of the invention
The technical problem to be solved in the present invention is to provide optimization process period, suitable concentration and the processing time of colchicin in wild jujube indefinite bud approach induction polyploid, is difficult to obtain isozygotying tetraploid problem to solve existing method.
For solving the problems of the technologies described above, the embodiment of the present invention provides a kind of method of wild jujube indefinite bud approach induction polyploid, comprise the steps: to start and cultivate, the young leaflet tablet of clip wild jujube aseptic seedling, carves and is inoculated into indefinite bud after wound and starts and carry out 9～11d pre-(secretly) cultivation on medium one; Induction is cultivated, and blade is gone in the liquid nutrient medium that contains 40～80mg/L colchicin and carries out multiploid induction cultivation, under dark condition, after 100rmp shaking table concussion cultivation 2～3d, proceeds on indefinite bud startup medium one and continues the dark 8～10d of cultivation; Extend and cultivate, pre-(secretly) is transferred to blade on Elongation of adventitious bud medium two after cultivating and finishing, and moves under light and cultivates 25～35d; Strong seedling cultivation, the indefinite bud that leaf regeneration is gone out is peeled off from blade, is transferred in the MS strong seedling culture base that contains plant growth regulator, cultivates 18～20d; Spire detects, and after indefinite bud robust growth, gets young leaflet tablet, carries out polyploid detection.
Wherein, described indefinite bud starts the plant growth regulator that medium one comprises the indole-3-butyric acid of improvement WPM minimal medium and the Thidiazuron that comprises 1.0mg/L and 0.3mg/L; Described Elongation of adventitious bud medium two comprises the plant growth regulator of the gibberellin of improvement WPM minimal medium and the indole-3-acetic acid that comprises 0.1mg/L and 0.5mg/L.
Wherein, described improvement WPM minimal medium contains sucrose 30g/L, ph=5.8～6.2, and filling a prescription is:
Wherein, described MS strong seedling culture base contains sucrose 30g/L, ph=5.8-6.2, and filling a prescription is:
Wherein, the 6-benzyl aminoadenine that the plant growth regulator in described strong seedling culture base is 1.0mg/L and the indolebutyric acid of 0.4mg/L.
Wherein, described young leaflet tablet is three pieces of blades of 3-5 piece that wild jujube aseptic seedling plant forms is learned upper end to lower end; Described blade is carved and is hindered method for 2～3 wounds of arteries and veins vertical direction crosscut in edge, cuts off master pulse but does not cut off blade; Described vaccination ways is proximal ends contact medium.
Wherein, it is not add the indefinite bud of agar to start medium one that liquid nutrient medium used is cultivated in described induction, and the concentration of described colchicin is 40～80mg/L.
Wherein, described elongation cultivation stage adopts manually operated condition of culture, and in described manually operated condition of culture, intensity of illumination is 19000x～21002x, light application time 12～16h/d, 23～27 DEG C of temperature.
Wherein, described polyploid detection method is flow cytometer method, and lysate used is improvement WPB lysate, the DAPI dye liquor that dye liquor used is 10ug/L.
Wherein, described improvement WPB lysate ph=7.2～7.8, filling a prescription is:
| Improvement WPB lysate composition | Configuration 1L content |
| Tris·HCl | 24.22g |
| MgCl 2·6H 2O | 0.81g |
| Na 2EDTA·2H 2O | 0.74g |
| NaCl | 5.03g |
| KCl | 5.96g |
| Na 2S 2O 5 | 1.90g |
| TritonX-100 | 10ml |
| PVP-10 | 10g |
The beneficial effect of technique scheme of the present invention is as follows:
In such scheme, by adopting the young leaflet tablet of wild jujube aseptic seedling as test material, can effectively overcome chimeric appearance, the efficiency that obtains homogenous polyploids plant is higher, and the genetic improvement of jujube tree is had to important value.
Brief description of the drawings
Fig. 1 is the young leaflet tablet of the wild jujube aseptic seedling after hindering the quarter of the embodiment of the present invention;
Fig. 2 is the schematic diagram of the aseptic young leaflet tablet of the wild jujube of the embodiment of the present invention after Elongation of adventitious bud medium two illumination cultivation 30d;
Fig. 3 is the schematic diagram after the wild jujube blade regenerated adventitious bud strong seedling culture 20d of the embodiment of the present invention;
Fig. 4 A is the wild jujube liploid plant DNA content schematic diagram of the embodiment of the present invention;
Fig. 4 B is the wild jujube tetraploid plant DNA content schematic diagram of the embodiment of the present invention;
Fig. 5 A is wild jujube liploid plant stomatal frequency and the large logotype of the embodiment of the present invention;
Fig. 5 B is wild jujube tetraploid plant stomatal frequency and the large logotype of the embodiment of the present invention;
Fig. 6 is the upgrowth situation schematic diagram of the wild jujube tetraploid plant of the embodiment of the present invention.
Embodiment
For making the technical problem to be solved in the present invention, technical scheme and advantage clearer, be described in detail below in conjunction with the accompanying drawings and the specific embodiments.
The embodiment of the present invention is difficult to obtain isozygotying tetraploid problem for existing method, and a kind of method of wild jujube indefinite bud approach induction polyploid is provided, and comprises the steps:
Start and cultivate, the young leaflet tablet of clip wild jujube aseptic seedling, carves and is inoculated into indefinite bud after wound and starts and carry out 9～11d pre-(secretly) cultivation on medium one;
Induction is cultivated, and blade is gone in the liquid nutrient medium that contains 40～80mg/L colchicin and carries out multiploid induction cultivation, under dark condition, after 100rmp shaking table concussion cultivation 2～3d, proceeds on indefinite bud startup medium one and continues the dark 8～10d of cultivation;
Extend and cultivate, pre-(secretly) is transferred to blade on Elongation of adventitious bud medium two after cultivating and finishing, and moves under light and cultivates 25～35d;
Strong seedling cultivation, the indefinite bud that leaf regeneration is gone out is peeled off from blade, is transferred in the MS strong seedling culture base (Murashige and Skoog, 1962) that contains plant growth regulator, cultivates 18～20d;
Spire detects, and after indefinite bud robust growth, gets young leaflet tablet, carries out polyploid detection.
By adopting the young leaflet tablet of wild jujube aseptic seedling as test material, can effectively overcome chimeric appearance, the efficiency that obtains homogenous polyploids plant is higher, and the genetic improvement of jujube tree is had to important value.
Particularly, the method for described wild jujube indefinite bud approach induction polyploid, comprises the steps:
Step 1, startup are cultivated
Prepare indefinite bud and start medium one, comprise the plant growth regulator of the indole-3-butyric acid of improvement WPM minimal medium (Woody Plant medium) and the Thidiazuron that comprises 1.0mg/L and 0.3mg/L, wherein said improvement WPM minimal medium is containing sucrose 30g/L, ph=6.0, filling a prescription is:
Choose wild jujube aseptic seedling plant forms and learn three pieces of blades of 3-5 piece of upper end to lower end, as shown in Figure 1,2 wounds of arteries and veins vertical direction crosscut in edge on every piece of blade, cut off master pulse but do not cut off blade, adopt proximal ends contact culture medium inoculated to start medium one at indefinite bud, inoculate altogether 144 pieces of blades, carry out 10d pre-(secretly) and cultivate.
Step 2, induction is cultivated
The liquid nutrient medium that preparation contains 75mg/L colchicin adds 75mg/L colchicin in the indefinite bud that does not add agar starts medium one.
Cultivate through 10d pre-(secretly), the petiole of blade and wound master pulse place have all grown the white point of similar callus, now for colchicin induction doubles to process best period, blade is proceeded to and in the liquid nutrient medium that contains 75mg/L colchicin, carry out multiploid induction cultivation, under dark condition, 3d is cultivated in the concussion of 100rmp shaking table, afterwards, proceed to indefinite bud and start continuation pre-(secretly) cultivation 10d on medium one.
Step 3, extends and cultivates
Prepare Elongation of adventitious bud medium two, comprise the plant growth regulator of the gibberellin of improvement WPM minimal medium and the indole-3-acetic acid that comprises 0.1mg/L and 0.5mg/L, described improvement WPM minimal medium formula is the same.
As shown in Figure 2, pre-(secretly) is transferred to blade on Elongation of adventitious bud medium two after cultivating and finishing, and move under light and cultivate, adopting manually operated condition of culture, in described manually operated condition of culture, intensity of illumination is 20000x, light application time 14h/d, 25 DEG C of temperature.
After 30d, have 120 pieces of blades and induce indefinite bud, average every piece of leaf regeneration bud number 2.29.Adventitious shoot regeneration rate is 83.33%, wherein adventitious shoot regeneration rate=induce indefinite bud blade number × 100%/inoculation explant number.
Step 4, strong seedling culture
Preparation MS strong seedling culture base, described MS strong seedling culture base contains sucrose 30g/L, ph=6.0, filling a prescription is:
And add the plant growth regulator of the 6-benzyl aminoadenine of 1.0mg/L and the indolebutyric acid of 0.4mg/L.
As shown in Figure 3, the indefinite bud that grows to 2cm is carefully peeled off from blade, and be inoculated on MS medium through being extended cultivation, inoculate altogether 275 indefinite buds, after cultivating 20d, have 211 indefinite buds to survive, survival rate is 76.72%.
Step 5, spire detects
The DAPI dye liquor of preparation improvement WPB lysate and 10ug/L, described improvement WPB lysate ph=7.5, filling a prescription is:
| Improvement WPB lysate composition | Configuration 1L content |
| Tris·HCl | 24.22g |
| MgCl 2·6H 2O | 0.81g |
| Na 2EDTA·2H 2O | 0.74g |
| NaCl | 5.03g |
| KCl | 5.96g |
| Na 2S 2O 5 | 1.90g |
| TritonX-100 | 10ml |
| PVP-10 | 10g |
As shown in Fig. 4 A to Fig. 6, strong seedling culture gained blade is shredded in containing in the culture dish of 1ml WPB lysate, use 200 order nylon net filters to centrifuge tube, add 100ul DAPI dye liquor, adopt cell instrument method to carry out polyploid detection.Finally obtain 14 tetraploid plants that isozygoty, tetraploid induction rate is 6.63%.
The above is the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, do not departing under the prerequisite of principle of the present invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (9)
1. a method for wild jujube indefinite bud approach induction polyploid, is characterized in that, comprises the steps:
Start and cultivate, the young leaflet tablet of clip wild jujube aseptic seedling, carves and is inoculated into indefinite bud after wound and starts and carry out 9～11d pre-(secretly) cultivation on medium one;
Induction is cultivated, and blade is gone in the liquid nutrient medium that contains 40～80mg/L colchicin and carries out multiploid induction cultivation, under dark condition, after 100rmp shaking table concussion cultivation 2～3d, proceeds on indefinite bud startup medium one and continues the dark 8～10d of cultivation;
Extend and cultivate, pre-(secretly) is transferred to blade on Elongation of adventitious bud medium two after cultivating and finishing, and moves under light and cultivates 25～35d;
Strong seedling cultivation, the indefinite bud that leaf regeneration is gone out is peeled off from blade, is transferred in the MS strong seedling culture base that contains plant growth regulator, cultivates 18～20d;
Spire detects, and after indefinite bud robust growth, gets young leaflet tablet, carries out polyploid detection.
2. the method for wild jujube indefinite bud approach induction polyploid according to claim 1, it is characterized in that, described indefinite bud starts the plant growth regulator that medium one comprises the indole-3-butyric acid of improvement WPM minimal medium and the Thidiazuron that comprises 1.0mg/L and 0.3mg/L;
Described Elongation of adventitious bud medium two comprises the plant growth regulator of the gibberellin of improvement WPM minimal medium and the indole-3-acetic acid that comprises 0.1mg/L and 0.5mg/L.
3. the method for wild jujube indefinite bud approach induction polyploid according to claim 2, is characterized in that, described improvement WPM minimal medium contains sucrose 30g/L, ph=5.8～6.2, and filling a prescription is:
4. the method for wild jujube indefinite bud approach induction polyploid according to claim 1, is characterized in that, described MS strong seedling culture base contains sucrose 30g/L, ph=5.8-6.2, and filling a prescription is:
5. the method for wild jujube indefinite bud approach induction polyploid according to claim 4, is characterized in that, the 6-benzyl aminoadenine that the plant growth regulator in described MS strong seedling culture base is 1.0mg/L and the indolebutyric acid of 0.4mg/L.
6. the method for wild jujube indefinite bud approach induction polyploid according to claim 1, is characterized in that, described young leaflet tablet is three pieces of blades of 3-5 piece that wild jujube aseptic seedling plant forms is learned upper end to lower end; Described blade is carved and is hindered method for 2～3 wounds of arteries and veins vertical direction crosscut in edge, cuts off master pulse but does not cut off blade; Described vaccination ways is proximal ends contact medium.
7. the method for wild jujube indefinite bud approach induction polyploid according to claim 1, is characterized in that, it is not add the indefinite bud of agar to start medium one that liquid nutrient medium used is cultivated in described induction, and the concentration of described colchicin is 40～80mg/L.
8. the method for wild jujube indefinite bud approach induction polyploid according to claim 1, it is characterized in that, described elongation cultivation stage adopts manually operated condition of culture, in described manually operated condition of culture, intensity of illumination is 19000x～21002x, light application time 12～16h/d, 23～27 DEG C of temperature.
9. the method for wild jujube indefinite bud approach induction polyploid according to claim 1, is characterized in that, described polyploid detection method is flow cytometer method, and lysate used is improvement WPB lysate, the DAPI dye liquor that dye liquor used is 10ug/L;
Wherein, described improvement WPB lysate ph=7.2～7.8, filling a prescription is:
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| CN108719046A (en) * | 2017-04-13 | 2018-11-02 | 北京林业大学 | A kind of method of induction culturing hybrid sweetgum tetraploid |
| CN112592935A (en) * | 2020-12-29 | 2021-04-02 | 安徽农业大学 | Genetic transformation method taking wild jujube callus as receptor |
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| CN112592935A (en) * | 2020-12-29 | 2021-04-02 | 安徽农业大学 | Genetic transformation method taking wild jujube callus as receptor |
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