CN102812903A - Method for germplasm innovation of chimera color-leafed plant through shoot tip cell layer rearrangement - Google Patents
Method for germplasm innovation of chimera color-leafed plant through shoot tip cell layer rearrangement Download PDFInfo
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- CN102812903A CN102812903A CN201210237049XA CN201210237049A CN102812903A CN 102812903 A CN102812903 A CN 102812903A CN 201210237049X A CN201210237049X A CN 201210237049XA CN 201210237049 A CN201210237049 A CN 201210237049A CN 102812903 A CN102812903 A CN 102812903A
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Abstract
The invention provides a method for germplasm innovation of a chimera color-leafed plant through shoot tip cell layer rearrangement. The method comprises the following steps of: selecting apical bud or axillalry bud of a parent chimera plant with chimerism as an explant, carrying out surface sterilization and flushing, inoculating on an initial culture medium for culture, establishing a rapid propagation system of axillalry bud or cluster bud of parent chimera, carrying out adventitious bud induction by the utilization of tissues such as stems or blades of parent chimera so as to generate a new type of chimeric bud which is different from parent chimera shoot tip in cell layer distribution structure and properties, screening novel chimera lines with excellent and stable properties for rooting induction and hardening-seedling, shifting to the field after survival, and screening so as to obtain a new species of color-leafed chimera. The invention has the following beneficial effects: the method has strong operationality, is simple and practical, requires a short breeding cycle, and can be widely applied in seed selection of the new species of the chimera color-leafed plant.
Description
Technical field
The present invention relates to the stripped rearrangement technical field of stem apex cell tier, especially a kind of method of chimeric sports lottery ticket leaf plant being carried out germplasm innovation of resetting through the stem apex cell tier.
Background technology
Color leaf plant is meant that blade presents the shades of colour except that greenery or comprises the plant of the multiple color of green.Because it is lucuriant in design that color leaf plant has, viewing period is long, is prone to characteristics such as maintenance, has become the new lover of afforestation, and the market demand is big.Wherein, Chimeric sports lottery ticket leaf plant is that color leaf plant is an important type colony (the chimera plant is that its apical meristem comprises two or more genotype cell and forms organ and the final whole plant that forms of growing jointly by these cell tiers); For example common golden heart bracketplant, " France " jade hairpin, double-colored India canna etc. all belong to chimeric sports lottery ticket leaf plant, and ornamental value is high.
Yet existing most of chimeric sports lottery ticket leaf plants all are spontaneous generations under field conditions (factors), and the difficulty of artificial synthetic technology is bigger; In addition, China chimeric sports lottery ticket leaf ornamental plants kind overwhelming majority is by external introduction, and the breadth and depth of autonomous innovation is all very limited.As everyone knows, improved seeds are keys that production high-quality flower plant and industry promote.Therefore, effectively utilize existing chimeric sports lottery ticket leaf plant resources to carry out germplasm innovation work, have great importance for the gap that strengthens China's flower industry international competitiveness, further with international breeding level fast.
Because most of angiospermous female and male gametophytes all derive from the L2 layer of its shoot apical meristem; Conventional sexual hybridization breeding meeting causes the separation of the unique proterties of chimeric sports lottery ticket leaf plant; The development that has greatly limited the plant breeding of chimeric sports lottery ticket leaf is (referring to Opportunities for synthetic plant chimeral breeding:Past and future.Burge GK; Morgan ER, Seelye JF.Plant Cell Tiss Organ Cult, the 70th the 1st phase of volume; The 13-21 page or leaf, 2002).Therefore, be badly in need of germplasm improvement and the innovative approach of a cover at present, and the stripped rearrangement technology of stem apex cell tier can filter out valuable chimera newtype from existing chimera plant to the chimera plant.But also not about resetting the technical method that chimeric sports lottery ticket leaf plant is carried out germplasm innovation, more there is not successful examples before this through the stem apex cell tier.
Summary of the invention
The object of the invention will solve the deficiency that above-mentioned technology exists just, and a kind of method of chimeric sports lottery ticket leaf plant being carried out germplasm innovation of resetting through the stem apex cell tier is provided.
The present invention solves the technical scheme that its technical problem adopts: thisly reset the method for chimeric sports lottery ticket leaf plant being carried out germplasm innovation through the stem apex cell tier, comprise the steps:
(1) the stem section of choosing the terminal bud of the chimera plant material with chimeric proterties or having axillalry bud carries out on superclean bench, washing 3 ~ 5 times with sterile distilled water after surface sterilization is handled to explant as explant;
(2) explant after will washing is inoculated on the initial culture base after inhaling the branch that anhydrates on the sterilization filter paper and cultivates, and cultivation temperature is that 25 ± 2 ° of C, intensity of illumination are 0～80 μ molm
-2S
-1, light application time is 8～12 hours/day;
The aseptic maternal chimera seedling that (3) will obtain is expanded numerous with the axillalry bud or the bud mode of growing thickly on proliferated culture medium;
(4) cut maternal chimeric stem section or leaf tissue and be inoculated in and carry out inducing of indefinite bud on the inducing culture, maternal chimera stem apex cell tier structure is arranged again, and then produce the chimeric bud of newtype that is different from maternal character;
(5) the chimeric bud of treating newtype is transferred to and carries out axillalry bud or the bud mode of growing thickly on the proliferated culture medium and expand numerously, and observes the stability of its proterties;
(6) filter out stable new chimera type strain system, and its plantlet is transferred to carries out culture of rootage on the root media;
(7) seedling after will taking root is taken out from medium; Wash the medium of root off and with seedling replanting to the nutritive cube that matrix is housed; Behind the refining seedling with seedling replanting to the field, treat that the plant back of become living further observes the stability of tree characteristics, screening obtains the chimera kind of newtype.
As preferably, described to reset the vegetable material that carries out germplasm innovation through the stem apex cell tier be chimera, is periclinal chimarea, sectorial chimera or mixochimaera type.
As preferably; In said step (2) (3) (4) (5); Described initial culture base, proliferated culture medium and inducing culture all are to be minimal medium with MS; Add 30g/L sucrose, 0.7% agar powder, 0.1 ~ 5mg/L basic element of cell division and 0.05 ~ 0.5mg/L growth hormone respectively, pH=5.8.
As preferably, red reticulate pattern grass is carried out the stem apex cell tier reset employed initial culture based formulas and be: MS+3% sucrose+0.7% agar+0.2mg/L6-BA+0.05mg/LNAA; Proliferated culture medium is: MS+3% sucrose+0.7% agar+0.5mg/L6-BA+0.1mg/LNAA; Inducing culture is: MS+3% sucrose+0.7% agar+3.0mg/L6-BA+0.2mg/L NAA.
As preferably, Wo Niujin is carried out the stem apex cell tier reset employed initial culture based formulas and be: MS+3% sucrose+0.7% agar+4.0mg/L6-BA+0.1mg/LIBA; Proliferated culture medium is: MS+3% sucrose+0.7% agar+1.0mg/L 6-BA+0.1mg/LNAA; Inducing culture is: MS+3% sucrose+0.7% agar+0.5mg/L6-BA+0.1mg/L NAA.
As preferably, in said step (4), the rearrangement of described maternal chimera stem apex cell tier structure realizes through inducing tissues such as its stem section or blade to produce chimeric indefinite bud.
As preferably; In said step (5) (6) (7), the plant that the acquisition of said newtype chimera kind need be passed through cultivation and field planting in the bottle carries out the chimera strain system that plant trait is observed and therefrom selected chimeric proterties can stablize maintenance and can survive.
As preferably, in said step (6), the prescription of said root media is 1/2MS+3% sucrose+0.8% agar+0.05 ~ 0.5mg/L growth hormone.
As preferably, in said step (7), said matrix is vermiculite and perlite, and vermiculite and perlitic volume ratio are 2:1.
As preferably, when in step (7), refining seedling, cover film and shading on seedling is opened film gradually at the refining seedling and is made seedling adapt to external environment after 6～12 days earlier.
The effect that the present invention is useful is: the present invention adopts the stripped rearrangement of stem apex cell tier that chimeric sports lottery ticket leaf plant is carried out germplasm innovation, has overcome the constraint of traditional breeding mode; And do not receive Effect of Environmental such as seasonal climate, but carry out in the anniversary; Breeding cycle is short, and acquisition can directly be carried out asexual fast numerous back application after stablizing the newtype chimera.This method is workable, and is simple and practical, and breeding efficiency is high, can be widely used in the breeding of new variety of chimeric sports lottery ticket leaf plant, has great importance for the utilization and the innovation of color leaf ornamental plants germ plasm resource.
Embodiment
Below in conjunction with embodiment the present invention is described further:
Embodiment 1:
Material: this instance is a material with the red reticulate pattern grass of periclinal chimarea plant (Fittonia verschaffeltii).
Step (1): draw materials and sterilize
The stem section of choosing the reticulate pattern grass is in order to keep the maternal chimeric characteristic of viewing and admiring as explant material.Explant earlier with the running water flushing, was used 75% alcohol disinfecting 30 seconds then, and using available chlorine content again is that 1% liquor natrii hypochloritis soaked 7 minutes, shake therebetween several times with thorough disinfection, at last on superclean bench with sterile distilled water flushing 4 times.
Step (2): inoculation
Material after the flushing is inoculated on the initial culture base after inhaling the branch that anhydrates on the sterilization filter paper cultivates, filling a prescription is MS+3% sucrose+0.7% agar+0.2mg/L6-BA+0.05mg/L NAA, pH=5.8.Condition of culture is 25 ± 2 ° of C, 40 μ mol m
-2s
-1, light application time is 16 hours/day.
Step (3): propagation
The aseptic maternal reticulate pattern grass that obtains is transferred to expands numerously on the proliferated culture medium with the bud mode of growing thickly, purpose is to keep under the prerequisite of maternal character colony being enlarged.The enrichment culture based formulas is: MS+3% sucrose+0.7% agar+0.5mg/L6-BA+0.1mg/LNAA, pH=5.8.Condition of culture is 25 ± 2 ° of C, 40 μ molm
-2s
-1, light application time is 16 hours/day.
Step (4): the rearrangement of stem apex cell tier structure
The blade of maternal reticulate pattern grass and stem section be cut into respectively to be inoculated in behind the square fritter in the 0.5cm left and right sides and the segment about 0.2cm carry out inducing of indefinite bud on the inducing culture, make generation be different from the chimeric bud of newtype of sharp cell tier arrangement of maternal reticulate pattern grass blade and proterties.The prescription of inducing culture is: MS+3% sucrose+0.7% agar+3.0mg/L6-BA+0.2mg/L NAA, pH=5.8.Condition of culture is 25 ± 2 ° of C, 40 μ mol m
-2s
-1, light application time is 16 hours/day.
Step (5): the screening of chimeric bud
The chimeric bud of the reticulate pattern of newtype grass transferred on the proliferated culture medium breed, observe the stability of all types of chimeric proterties, and then to filter out stable new chimera type strain be 4.The enrichment culture based formulas is: MS+3% sucrose+0.7% agar+0.5mg/L6-BA+0.2mg/LNAA, pH=5.8.Condition of culture is 25 ± 2 ° of C, 40 μ mol m
-2s
-1, light application time is 16 hours/day.
Step (6): take root
The newtype reticulate pattern that filters out grass chimera is transferred to carries out root media on the root media, filling a prescription is 1/2MS+3% sucrose+0.7% agar+0.1mg/L NAA (NAA).Condition of culture: temperature is 25 ± 2 ° of C, 80 μ molm
-2s
-1Illumination is cultivated down, and light application time is 12 hours/day.
Step (7): the transplanting of seedling and the acquisition of new varieties
After seedling takes root, it is taken out from blake bottle; Rinse out the medium of root gently, transplant then that (matrix in the nutritive cube is vermiculite and perlite, v/v=2:1) to nutritive cube; Wilt for preventing the seedling dehydration; Improve transplanting survival rate, can above seedling, also suitably shade to keep humidity by cover film, begin to open gradually film after 2 days and make seedling adapt to external environment.Approximately the refining seedling moved to the field after 10 days, thereby obtained 1 the reticulate pattern grass chimera new varieties that can under field condition, survive.
Embodiment 2:
Material: this instance is a material with Wo Niujin (Gasteriea armstrongii f.variegata).
Step (1): draw materials and sterilize
Choose the bright and beautiful scape of the ox for sleeping in that has yellow and green simultaneously as explant material, reason is with embodiment 1.Unopened bud on the inflorescence is downcut; Earlier with the running water flushing, used 75% alcohol disinfecting then 45 seconds, using available chlorine content again is that 1% liquor natrii hypochloritis soaked 6 minutes; Shake therebetween several times with thorough disinfection, at last on superclean bench with sterile distilled water flushing 4 times.
Step (2): inoculation
Material after the flushing is inoculated on the initial culture base after inhaling the branch that anhydrates on the sterilization filter paper cultivates, filling a prescription is MS+3% sucrose+0.7% agar+4.0mg/L6-BA+0.1mg/LIBA (indolebutyric acid), pH=5.8.Condition of culture is 25 ± 2 ° of C, the dark cultivation.
Step (3): the rearrangement of propagation and stem apex cell tier structure
Treat after callus forms material transfer to be continued to cultivate down to illumination condition; The callus that will change green differentiation subsequently is transferred to and carries out inducing of indefinite bud on the inducing culture; Make the chimeric bud of Wo Niujin that produces different stem apex cell tier arrangements and proterties type, thereby accomplish the rearrangement of stem apex cell tier arrangement.The inducing culture based formulas is: MS+3% sucrose+0.7% agar+0.5mg/L6-BA+0.1mg/LNAA, pH=5.8.Condition of culture is 25 ± 2 ° of C, 40 μ mol m
-2S-
1, light application time is 16 hours/day.
Step (4): the screening of chimeric bud
The chimeric bud of the Wo Niujin of newtype transferred on the proliferated culture medium breed, observe the stability of all types of chimeric proterties, and then filter out stable new chimera type strain system.The enrichment culture based formulas is: MS+3% sucrose+0.7% agar+1.0mg/L6-BA+0.1mg/L NAA, pH=5.8.Condition of culture is 25 ± 2 ° of C, 40 μ mol m
-2s
-1, light application time is 16 hours/day.
Step (5): take root
The newtype that the filters out ox brocade chimera that crouches is transferred to and carries out root media on the root media, and filling a prescription is 1/2MS+3% sucrose+0.7% agar+0.1mg/L NAA (NAA).Condition of culture: temperature is 25 ± 2 ° of C, 80 μ molm
-2s
-1Illumination is cultivated down, and light application time is 12 hours/day.
Step (6): the transplanting of seedling and the acquisition of new varieties
After seedling takes root, it is taken out from blake bottle; Rinse out the medium of root gently;, the shady and cool ventilation place transplants that (matrix is vermiculite and perlite to nutritive cube after placing 3 ~ 5 days; V/v=2:1), the ox brocade plant thereby the chimera that obtains newtype of the present invention crouches, transplanting survival rate can reach 100%.
Need to prove that at last what more than enumerate only is practical implementation example of the present invention.Obviously, the invention is not restricted to above examples of implementation, many distortion can also be arranged.All distortion that those of ordinary skill in the art can directly derive or associate from content disclosed by the invention all should be thought protection scope of the present invention.
Claims (10)
1. reset the method for chimeric sports lottery ticket leaf plant being carried out germplasm innovation through the stem apex cell tier for one kind, it is characterized in that comprising the steps:
(1) the stem section of choosing the terminal bud of the chimera plant material with chimeric proterties or having axillalry bud carries out on superclean bench, washing 3 ~ 5 times with sterile distilled water after surface sterilization is handled to explant as explant;
(2) explant after will washing is inoculated on the initial culture base after inhaling the branch that anhydrates on the sterilization filter paper and cultivates, and cultivation temperature is that 25 ± 2 ° of C, intensity of illumination are 0～80 μ molm
-2S
-1, light application time is 8～12 hours/day;
The aseptic maternal chimera seedling that (3) will obtain is expanded numerous with the axillalry bud or the bud mode of growing thickly on proliferated culture medium;
(4) cut maternal chimeric stem section or leaf tissue and be inoculated in and carry out inducing of indefinite bud on the inducing culture, maternal chimera stem apex cell tier structure is arranged again, and then produce the chimeric bud of newtype that is different from maternal character;
(5) the chimeric bud of treating newtype is transferred to and carries out axillalry bud or the bud mode of growing thickly on the proliferated culture medium and expand numerously, and observes the stability of its proterties;
(6) filter out stable new chimera type strain system, and its plantlet is transferred to carries out culture of rootage on the root media;
(7) seedling after will taking root is taken out from medium; Wash the medium of root off and with seedling replanting to the nutritive cube that matrix is housed; Behind the refining seedling with seedling replanting to the field, treat that the plant back of become living further observes the stability of tree characteristics, screening obtains the chimera kind of newtype.
2. the method for chimeric sports lottery ticket leaf plant being carried out germplasm innovation of resetting through the stem apex cell tier according to claim 1; It is characterized in that: described vegetable material through stem apex cell tier rearrangement carrying out germplasm innovation is a chimera, is periclinal chimarea, sectorial chimera or mixochimaera type.
3. the method for chimeric sports lottery ticket leaf plant being carried out germplasm innovation of resetting through the stem apex cell tier according to claim 1; It is characterized in that: in said step (2) (3) (4) (5); Described initial culture base, proliferated culture medium and inducing culture all are to be minimal medium with MS; Add 30g/L sucrose, 0.7% agar powder, 0.1 ~ 5mg/L basic element of cell division and 0.05 ~ 0.5mg/L growth hormone respectively, pH=5.8.
4. according to claim 3ly reset the method for chimeric sports lottery ticket leaf plant being carried out germplasm innovation, it is characterized in that: red reticulate pattern grass is carried out the stem apex cell tier reset employed initial culture based formulas and be: MS+3% sucrose+0.7% agar+0.2mg/L6-BA+0.05mg/LNAA through the stem apex cell tier; Proliferated culture medium is: MS+3% sucrose+0.7% agar+0.5mg/L6-BA+0.1mg/LNAA; Inducing culture is: MS+3% sucrose+0.7% agar+3.0mg/L6-BA+0.2mg/LNAA.
5. according to claim 3ly reset the method for chimeric sports lottery ticket leaf plant being carried out germplasm innovation, it is characterized in that: Wo Niujin is carried out the stem apex cell tier reset employed initial culture based formulas and be: MS+3% sucrose+0.7% agar+4.0mg/L6-BA+0.1mg/LIBA through the stem apex cell tier; Proliferated culture medium is: MS+3% sucrose+0.7% agar+1.0mg/L6-BA+0.1mg/LNAA; Inducing culture is: MS+3% sucrose+0.7% agar+0.5mg/L6-BA+0.1mg/LNAA.
6. stem apex cell tier according to claim 1 is reset the method for chimeric sports lottery ticket leaf plant being carried out germplasm innovation; It is characterized in that: in said step (4); The rearrangement of described maternal chimera stem apex cell tier structure realizes through inducing tissues such as its stem section or blade to produce chimeric indefinite bud.
7. the method for chimeric sports lottery ticket leaf plant being carried out germplasm innovation of resetting through the stem apex cell tier according to claim 1; It is characterized in that: in said step (5) (6) (7), the plant that the acquisition of said newtype chimera kind need be passed through cultivation and field planting in the bottle carries out the chimera strain system that plant trait is observed and therefrom selected chimeric proterties can stablize maintenance and can survive.
8. stem apex cell tier according to claim 1 is reset the method for chimeric sports lottery ticket leaf plant being carried out germplasm innovation; It is characterized in that: in said step (6), the prescription of said root media is 1/2MS+3% sucrose+0.8% agar+0.05 ~ 0.5mg/L growth hormone.
9. according to claim 1ly reset the method for chimeric sports lottery ticket leaf plant being carried out germplasm innovation through the stem apex cell tier, it is characterized in that: in said step (7), said matrix is vermiculite and perlite, and vermiculite and perlitic volume ratio are 2:1.
10. the method for chimeric sports lottery ticket leaf plant being carried out germplasm innovation of resetting through the stem apex cell tier according to claim 1; It is characterized in that: when in step (7), refining seedling; Earlier cover film and shading on seedling is opened film gradually at the refining seedling and is made seedling adapt to external environment after 6～12 days.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104957039A (en) * | 2015-07-10 | 2015-10-07 | 青岛华盛绿能农业科技有限公司 | Rapid propagation and maintenance method for fittonia verschaffeltii |
| CN114680046A (en) * | 2022-04-22 | 2022-07-01 | 海南茗卉农林科技发展有限公司 | Tissue culture rapid propagation method for keeping stable characters of color leaf chimera ornamental plants |
-
2012
- 2012-07-10 CN CN201210237049XA patent/CN102812903A/en active Pending
Non-Patent Citations (1)
| Title |
|---|
| 李雅志等: "《无性繁殖植物辐射育种新方法》", 31 May 1991, 原子能出版社 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104957039A (en) * | 2015-07-10 | 2015-10-07 | 青岛华盛绿能农业科技有限公司 | Rapid propagation and maintenance method for fittonia verschaffeltii |
| CN114680046A (en) * | 2022-04-22 | 2022-07-01 | 海南茗卉农林科技发展有限公司 | Tissue culture rapid propagation method for keeping stable characters of color leaf chimera ornamental plants |
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Application publication date: 20121212 |