CN109757370A - A kind of method that lagerstroemia fauriei Koehne tissue culture regeneration is established - Google Patents
A kind of method that lagerstroemia fauriei Koehne tissue culture regeneration is established Download PDFInfo
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- CN109757370A CN109757370A CN201711100467.3A CN201711100467A CN109757370A CN 109757370 A CN109757370 A CN 109757370A CN 201711100467 A CN201711100467 A CN 201711100467A CN 109757370 A CN109757370 A CN 109757370A
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Abstract
The present invention relates to a kind of methods that lagerstroemia fauriei Koehne tissue culture regeneration is established, this method is sprouted using lagerstroemia fauriei Koehne seed obtains sterile seedling, then clip young leaflet tablet is explant, by carrying out dedifferentiation culture to explant, the strong callus of differentiation capability again is obtained, and then carries out dedifferentiation culture and subsequent culture of rootage realization regeneration plant using callus.The present invention is explant using sterile seedling, avoid traditional explant due to differentiation degree is high and dedifferentiation is not easy the problem high with pollution rate, it is easy to operate, feasible.Specifically include following steps: the acquisition of 1. Aseptic seedlings；2. the induction of callus；3. the subculture and proliferation of callus；4. callus breaks up again；5. culture of rootage；6. plant regeneration.For the callus proliferation times of FuShi crape myrtle up to 2.1 times, tissue culture regeneration rate reaches 95% or more in the present invention, has established solid experiment basis for the foundation of crape myrtle genetic conversion system.
Description
Technical field
The present invention relates to a kind of methods that lagerstroemia fauriei Koehne tissue culture regeneration is established, and belong to woody plant tissure culture technique
Field.
Background technique
Crape myrtle (Lagerstroemia indica) belong to Lythraceae (Lythraceae) Lagerstroemia, alias " one hundred days
It is red ", " all round victory ".Crape myrtle is originating in China, is one of China's tradition famous flower.Domestic application mainly has banaba
(L.speciosa), Fujian crape myrtle (L.limii), Nan Ziwei (L.subcostata) etc., foreign countries cultivate also only big extensively
Spend crape myrtle and two kinds of crape myrtle.Crape myrtle is typically used as machaka or arbor plant, and since crape myrtle trunk is smooth, tree performance is graceful,
It is adaptable, it is cultivated frequently as shade tree, streetscape tree, some cities are using crape myrtle as city flower (tree).
In terms of breeding, breeding scholar in the U.S.'s carried out the breeding research of crape myrtle since 1962, was mixed with by planting interior, kind
Breeding is handed over, mildew-resistance and the red gorgeous kind of pattern are selected, Japanese Takii seedling Co., Ltd. utilizes imported from our country
Crape myrtle resource selects short life and spends more kind.In recent years, China also starts to make full use of domestic crape myrtle resource progress abundant miscellaneous
Breeding and relevant research are handed over, is expected to select the new varieties that a batch has ornamental value, is answered to preferably enrich crape myrtle gardens
Use kind.
Since traditional genetic breeding has biggish blindness, the disadvantages of breeding period is long, and modern cell engineering
The means such as breeding and molecular breeding overcome these disadvantages.In recent years, woody plant tissure culture studies obtain remarkable progress,
Such as poplar, plum blossom, eucalyptus, plane tree, regeneration can be completed by somatic embryogenesis pathway or adventitious organogenesis, but
It is extremely difficult from nutrition organs somatic embryos for xylophyta.Plant regeneration is set to be pair by the method for tissue cultures
Plant carries out the important prerequisite condition of transgene improvement, and current most of genetic conversion system requires to establish a stabilization
Plant regeneration system carry out the transfer of gene, the selection and regeneration of transformed plant, including direct or indirect regenerating system
Have been widely used for plant genetic improvement.
Summary of the invention
To lay the foundation to crape myrtle genetic transformation, the present invention provide a kind of lagerstroemia fauriei Koehne (big safflower crape myrtle:
Lagerstroemia × ' Tuscarora ') tissue culture regeneration establish method.
In order to achieve the above objectives, the present invention provides a kind of method of lagerstroemia fauriei Koehne tissue culture regeneration, specific steps are such as
It is lower described:
The present invention provides a kind of method of lagerstroemia fauriei Koehne tissue culture regeneration, it is described that specific step is as follows:
1) acquisition of Aseptic seedling
Well-developed seed on FuShi crape myrtle health elite stand is harvested, seed wing is manually removed and prevents damage kind skin, is carried out disinfection:30min first is rinsed with tap water, then 75% alcohol treatment 30s；1% sodium hypochlorite sterilizes 15min, then sterile water
It rinses 5 times, is seeded in after disinfection in MS minimal medium, obtains the seedling of robust growth；
2) induction of callus
20 days blades of sterile culture in clip step (1) under vertical vane master pulse cuts 3~4, keep side leaf margin to be connected,
Face of blade is inoculated in calli induction media 1/2MS+6-BA 1.0mg/L+2,4-D 1.0+sucrose 30g/L+ fine jade upward
In rouge 7g/L, dark culture processing 7 days, 25 DEG C of cultivation temperature or so, intensity of illumination 2000-3000lx, light application time 14h/d；
3) subculture and proliferation of callus
The callus obtained in step (2) is cut into diameter about 6-10mm and is inoculated in common B5 culture medium, every bottle of culture medium
5-10 pieces of callus are connect, dark culture, cultivation temperature is 23-27 DEG C, and every 20 days subcultures are primary, cultivates 20 days, obtains callus group
Knitting fresh weight and being proliferated is original 2.1 times；
4) differentiation of callus
The callus obtained in step (3) is inoculated in differential medium WPM+6-BA 1.0+IBA 0.2+Vc 100mg/L
Induction again, 2 pieces of callus of every bottle of inoculation are carried out in+sucrose 30g/L+ agar 7g/L, every processing is inoculated with 15 bottles respectively,
Cultivation temperature is 25 DEG C, intensity of illumination 2000-3000lx, light application time 14h/d；
5) culture of rootage
The unrooted test tube seedling containing adventitious bud obtained in step (4) is inoculated in root media improvement 1/2M+NAA 0.5
It being carried out culture of rootage 15-20 days in 0.5 mg/L+ sucrose 30g/L+ agar 7g/L of mg/L+IBA, cultivation temperature is 23 ± 3 DEG C,
Light application time is 14 h/d, intensity of illumination 1500-2000lx；
6) hardening of aseptic seedling
The culture bottle cap of the lagerstroemia fauriei Koehne band root aseptic seedling obtained in step (5) is opened, then indoor culture 5-7 days moves
It plants in the matrix using carbendazim disinfection, covers moisturizing using PVC film with holes, be placed on ventilating and cooling place and cultivate 25-30 days, training
It supports by the 15th day and removes PVC film, use the sunshade net of shading rate 75% -80% instead, cultivating matrix components used is volume ratio 1:
1 peat soil and perlite.
The method that the big safflower crape myrtle tissue culture regeneration of the present invention is established has the advantage that
1) regenerating system has the features such as high callus induction rate, simple Reproduction methods, inheritance stability, can be for as something lost
Pass the object of conversion.Therefore, this technology is that the foundation of lagerstroemia fauriei Koehne genetic conversion system lays the foundation；
2) callus induction is carried out using the blade of sterile seed seedling, power of regeneration is strong and not easy to pollute.Regeneration rate reaches
95% or more.
Specific embodiment
The present invention provides a kind of method of lagerstroemia fauriei Koehne tissue culture regeneration, it is described that specific step is as follows:
1) acquisition of Aseptic seedling
Well-developed seed on FuShi crape myrtle health elite stand is harvested, seed wing is manually removed and prevents damage kind skin, is carried out disinfection:30min first is rinsed with tap water, then 75% alcohol treatment 30s；1% sodium hypochlorite sterilizes 15min, then sterile water
It rinses 5 times, is seeded in after disinfection in MS minimal medium, obtains the seedling of robust growth；
2) induction of callus
20 days blades of sterile culture in clip step (1) under vertical vane master pulse cuts 3~4, keep side leaf margin to be connected,
Face of blade is inoculated in calli induction media 1/2MS+6-BA 1.0mg/L+2,4-D 1.0+sucrose 30g/L+ fine jade upward
In rouge 7g/L, dark culture processing 7 days, 25 DEG C of cultivation temperature or so, intensity of illumination 2000-3000lx, light application time 14h/d；
3) subculture and proliferation of callus
The callus obtained in step (2) is cut into diameter about 6-10mm and is inoculated in common B5 culture medium, every bottle of culture medium
5-10 pieces of callus are connect, dark culture, cultivation temperature is 23-27 DEG C, and every 20 days subcultures are primary, cultivates 20 days, obtains callus group
Knitting fresh weight and being proliferated is original 2.1 times；
4) differentiation of callus
The callus obtained in step (3) is inoculated in differential medium WPM+6-BA 1.0+IBA 0.2+Vc 100mg/L
Induction again, 2 pieces of callus of every bottle of inoculation are carried out in+sucrose 30g/L+ agar 7g/L, every processing is inoculated with 15 bottles respectively, training
Supporting temperature is 25 DEG C, intensity of illumination 2000-3000lx, light application time 14h/d, and callus differentiation rate is up to 95.7%；
5) culture of rootage
The unrooted test tube seedling containing adventitious bud obtained in step (4) is inoculated in root media improvement 1/2M+NAA 0.5
It being carried out culture of rootage 15-20 days in 0.5 mg/L+ sucrose 30g/L+ agar 7g/L of mg/L+IBA, cultivation temperature is 23 ± 3 DEG C,
Light application time is 14 h/d, intensity of illumination 1500-2000lx；
6) hardening of aseptic seedling
The culture bottle cap of the lagerstroemia fauriei Koehne band root aseptic seedling obtained in step (5) is opened, then indoor culture 5-7 days moves
It plants in the matrix using carbendazim disinfection, covers moisturizing using PVC film with holes, be placed on ventilating and cooling place and cultivate 25-30 days, training
It supports by the 15th day and removes PVC film, use the sunshade net of shading rate 75% -80% instead, cultivating matrix components used is volume ratio 1:
1 peat soil and perlite.
1-2cm of root long, 3-4 cm of height of seedling tissue-cultured seedling transplanted after indoor hardening 7 days, survival rate is up to 90%.
Above-mentioned induced medium, subculture multiplication medium contain agar 9.0g/L, 30 g/ of sucrose in root media
L, pH value are as follows: 5.8-6.0.
Big safflower crape myrtle (Lagerstroemia × ' Tuscarora ') tissue culture regeneration is carried out using the above method to build
It is vertical, the seedling of tissue culture containing root is successfully obtained, solid experiment basis has been established in the foundation of crape myrtle genetic conversion system.
Although disclosing the present invention above with preferred embodiment, so it is not intended to limiting the invention, all using equivalent
Replacement or equivalent transformation mode technical solution obtained, are within the scope of the present invention.
Claims (6)
1. the present invention provides a kind of methods of lagerstroemia fauriei Koehne tissue culture regeneration, which is characterized in that including the following steps:
The acquisition of Aseptic seedling
Well-developed seed on FuShi crape myrtle health elite stand is harvested, seed wing is manually removed and prevents damage kind skin, is carried out disinfection:30min first is rinsed with tap water, then 75% alcohol treatment 30s；1% sodium hypochlorite sterilizes 15min, then sterile water
It rinses 5 times, is seeded in after disinfection in MS minimal medium, obtains the seedling of robust growth；
The induction of callus
20 days blades of sterile culture in clip step (1) under vertical vane master pulse cuts 3~4, keep side leaf margin to be connected,
Face of blade is inoculated in calli induction media upward, dark culture processing 7 days, and 25 DEG C of cultivation temperature or so, intensity of illumination
2000-3000lx, light application time 14h/d；
The subculture and proliferation of callus
The callus obtained in step (2) is cut into diameter about 6-10mm and is inoculated in common B5 culture medium, every bottle of culture medium
5-10 pieces of callus are connect, dark culture, cultivation temperature is 23-27 DEG C, and every 20 days subcultures are primary, cultivates 20 days, obtains callus group
Knitting fresh weight and being proliferated is original 2.1 times；
The differentiation of callus
The callus obtained in step (3) is inoculated in differential medium and carries out induction again, 2 pieces of callus of every bottle of inoculation
Tissue, every processing are inoculated with 15 bottles respectively, and cultivation temperature is 25 DEG C, intensity of illumination 2000-3000lx, light application time 14h/d；
(5) the unrooted test tube seedling containing adventitious bud obtained in step (4) is inoculated in root media and carries out by culture of rootage
Culture of rootage 15-20 days, cultivation temperature was 23 ± 3 DEG C, and light application time is 14 h/d, intensity of illumination 1500-2000lx；
(6) hardening of aseptic seedling
The culture bottle cap of the lagerstroemia fauriei Koehne band root aseptic seedling obtained in step (5) is opened, then indoor culture 5-7 days moves
It plants in the matrix using carbendazim disinfection, covers moisturizing using PVC film with holes, be placed on ventilating and cooling place and cultivate 25-30 days, training
It supports by the 15th day and removes PVC film, use the sunshade net of shading rate 75% -80% instead, cultivating matrix components used is volume ratio 1:
1 peat soil and perlite.
2. the method for lagerstroemia fauriei Koehne tissue culture regeneration according to claim 1, it is characterised in that: in step (2)
Callus inducing medium is 1/2MS+6-BA 1.0mg/L+2,4-D 1.0+sucrose 30g/L+ agar 7g/L.
3. the method for lagerstroemia fauriei Koehne tissue culture regeneration according to claim 1, it is characterised in that: in step (3)
Callus subculture medium is common B5 medium.
4. the method for lagerstroemia fauriei Koehne tissue culture regeneration according to claim 1, it is characterised in that: in step (4)
It is WPM+6-BA 1.0+IBA 0.2+Vc 100mg/L+sucrose 30g/L+ fine jade that callus is differentiated to form adventitious bud culture base again
Rouge 7g/L.
5. the method for lagerstroemia fauriei Koehne tissue culture regeneration according to claim 1, it is characterised in that: in step (5)
Adventitious bud rooting culture medium is improvement 0.5 mg/L+IBA of 1/2M+NAA, 0.5 mg/L+ sucrose 30g/L+ agar 7g/L.
6. the method for lagerstroemia fauriei Koehne tissue culture regeneration according to claim 1, it is characterised in that: in step (1)
Seed sterilization method be first with tap water rinse 30min, then use 75% alcohol treatment 30s, then 1% sodium hypochlorite sterilizing
15min, last aseptic water washing 5 times.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112493126A (en) * | 2020-11-23 | 2021-03-16 | 河北科技师范学院 | Method for induction of lagerstroemia indica somatic embryo and plant regeneration |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112493126A (en) * | 2020-11-23 | 2021-03-16 | 河北科技师范学院 | Method for induction of lagerstroemia indica somatic embryo and plant regeneration |
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Application publication date: 20190517 |