CN106993533A - A kind of cultural method of cassava fragility embryo callus - Google Patents
A kind of cultural method of cassava fragility embryo callus Download PDFInfo
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Abstract
The invention discloses a kind of cultural method of cassava fragility embryo callus, comprise the following steps：(1) fragility embryonic callus induction；(2) fragility embryo callus regenerates.The cultural method of cassava fragility embryo callus embryonic callus induction generation time of enbrittling is short, the high advantage of generation rate, and cassava is preferably the conventional cultivation kinds such as south China 8 and south China 6.
Description
Technical field
The invention belongs to fragility embryo callus culture technique field, and in particular to a kind of cassava fragility embryo callus subculture group
The cultural method knitted.
Background technology
Cassava (Manihot esculenta Crantz) is Euphorbiaceae casava, is a kind of important grain and energy
Source plant.People generally improve its quality using traditional breeding method mode, but some characters such as Resistant, reduction cyanide contain
Amount etc. could only be improved by genetic engineering means.And an effective regenerating system is genetic engineering means successful implementation
Prerequisite.Fragility embryo callus is a kind of diameter≤1mm tissue, is easily disperseed during Liquid Culture, and propagation is fast,
Most cells have totipotency, it is possible thereby to the high-quality embryogenic suspension cultures obtained.Because it can increase outer
The probability for producing a large amount of transformed cells is integrated in source gene insertion, so being a kind of ideal genetic transformation destination organization.It is crisp
Property embryo callus be used to by particle bombardment, electrotransformation and agrobacterium-mediated transformation obtain transgenosis Manihot Esculenta.
The induction of cassava embryo callus is more complicated.The induction of fragility embryonal connective tissue is very big by genotype effect, and occurs frequency
Rate is very low, and induction time is long.Therefore a kind of method for inducing and cultivating of cassava fragility embryo callus rapidly and efficiently is set up,
It is significant to cassava transgenic breeding.
The content of the invention
It is an object of the invention to provide a kind of cultural method of cassava fragility embryo callus, this method is quickly high
Effect.
The above-mentioned purpose of the present invention is achieved through the following technical solutions：A kind of cassava fragility embryo callus
Cultural method, comprises the following steps：
(1) fragility embryonic callus induction
(1.1) cassava body embryo block is chosen, after pretreatment, dark training is placed on fragility embryonic callus induction culture medium
Support, induction produces nascent fragility embryo callus；
(1.2) nascent fragility embryo callus is transferred on fresh fragility embryonic callus induction culture medium and increased
Expansion culture is grown, fragility embryo callus is obtained；
(2) fragility embryo callus regenerates
(2.1) fragility embryo callus is collected into liquid bulk embryonal induction culture medium, after concussion and cultivate, it is unnecessary to remove
Soft non-embryonal connective tissue, then outwell liquid bulk embryonal induction culture medium, then fragility embryo callus is placed in body embryo induction
Continue Fiber differentiation in culture medium and produce body embryo；
(2.2) body embryo is transferred on fresh body embryonal induction culture medium, body embryo is carried out under dark condition to expand numerous culture, obtained
Body embryo after numerous culture must be expanded；
(2.3) body embryo after numerous culture will be expanded and be transferred to culture in body embryo maturation medium, promote body embryo maturation sub for green
Blade profile embryo；
(2.4) green cotyledon type embryo is transferred in body embryo germination medium and cultivated, promoted green cotyledon type embryo germination, enter
And seedling is regenerated, obtain Manihot Esculenta.
In the cultural method of above-mentioned cassava fragility embryo callus：
Pretreatment described in step (1.1) of the present invention includes cassava body embryo block being placed in the container containing mannitol, uses
Breathable sealing film is sealed, and then container is placed in vavuum pump, is adjusted and is started timing when air pressure drops to 0.08Mpa, 15~
Taken out after 20min, in the workbench sterilized, above-mentioned treated cassava body embryo block is taken out, water is blotted with sterilizing filter paper
After point, dark culturing 20~30 days on fragility embryonic callus induction culture medium are placed on, induction produces nascent fragility embryo
The concentration of callus, wherein mannitol is 250~300mmol/L.
It is preferred that, pretreatment described in step (1.1) of the present invention includes cassava body embryo block being placed in the appearance containing mannitol
In device, sealed, container be placed in vavuum pump with breathable sealing film then, regulation air pressure starts timing when dropping to 0.08Mpa,
Take out, in the workbench sterilized, above-mentioned treated body embryo block is taken out after 15min, after sterilizing filter paper suck dry moisture,
Dark culturing 30 days on fragility embryonic callus induction culture medium are placed on, induction produces nascent fragility embryo callus,
Wherein the concentration of mannitol is 300mmol/L.
Cassava body embryo block of the present invention first purified processing, purifying is that the non embryogenic callus around body embryo block is tried one's best clearly
Except clean.Cassava body embryo block is placed in mannitol to the effect with regulation body embryo block osmotic potential, regulation air pressure is vacuumized can be with
Body embryo block is fully contacted with mannitol.
Cassava body embryo block in the present invention is preferably derived from Manihot esculenta Crantz. cv. M. SC 6, it would however also be possible to employ cassava of south China 8 etc. is other
The cassava of kind.
Fragility embryonic callus induction culture medium (Friable in step (1.1)~(1.2) of the present invention
Embryogenic callus induction medium, FECIM) include following component：GD(Gresshof and Doy
Basal Medium), 2,4-D (2,4- dichlorphenoxyacetic acid), sucrose and agar powder, add in wherein 1L GD 2,4-D 8~
12mg, 15~20g of sucrose, 10~15g of agar powder.
It is preferred that, 1L fragility embryonic callus induction culture mediums (Friable in step (1.1)~(1.2) of the present invention
Embryogenic callus induction medium, FECIM) include following component：GD(Gresshof and Doy
Basal Medium), 2,4-D, sucrose and agar powder, add 2,4-D 8mg, sucrose 15g, agar powder 10g in wherein 1L GD.
Fragility embryonic callus induction culture medium effect of the present invention is that the dedifferentiation of induction body embryo produces fragility embryo callus subculture
Tissue.Embryo callus be it is loose, it is spherical, have embryo activity cell Youth League organization.Fragility embryonic callus induction culture
GD is that the nutritional ingredients such as basic N P and K calcium, molysite, trace element and organic matter are provided for tissue growth in base, and 2,4-D are
Promote the class auxin material of body embryo dedifferentiation.Sucrose provides carbon source for the growth of tissue.Agar powder has been to solidify culture medium
Effect.
Nascent fragility embryo callus is transferred to fragility embryonic callus induction culture in step (1.2) of the present invention
Propagation expands culture on base, and (being more preferably 2 weeks) subculture can once obtain a large amount of fragility embryo callus within 2~3 weeks.
Further, the fragility embryo callus can be used for transgenosis, protoplast extraction etc..
Body embryo inducing culture (somatic embryo induction medium, SEIM) in step (2.1) of the present invention
Including following component：NMS culture mediums (new Murashige and Skoog), 2,4-D, sucrose and agar powder, wherein 1L NMS
Being added in culture medium has 8~10mg of 2,4-D, 20~30g of sucrose, 7.5~8.5g of agar powder.
It is preferred that, 1L body embryos inducing culture (somatic embryo induction in step (2.1) of the present invention
Medium, SEIM) include following component：NMS culture mediums (new Murashige and Skoog), 2,4-D, sucrose and agar
Being added in powder, wherein 1L NMS culture mediums has 2,4-D 8mg, sucrose 25g, agar powder 7.5g.
Wherein NMS culture mediums are by CaCl in MS (Murashige and Skoog) culture medium2Consumption is expanded as original
5 times, other constant acquisitions of MS medium components, use preceding regulation pH to 6.0~6.2, autoclave sterilization 20min；Liquid
Body embryo inducing culture does not include agar powder.
In the present invention NMS for tissue growth provide the nutrition such as basic N P and K calcium, molysite, trace element and organic matter into
Point, 2,4-D be the class auxin material for promoting the differentiation of fragility embryo callus.Sucrose provides carbon source for the growth of tissue.Fine jade
Cosmetics is to play a part of to solidify culture medium.
Fragility embryo callus is collected into liquid bulk embryonal induction culture medium in step (2.1) of the present invention, concussion training
Support after 1~2h (1h is more preferably), remove unnecessary soft non-embryonal connective tissue, then outwell liquid bulk embryonal induction culture medium, then will be crisp
Property embryo callus be placed in body embryo inducing culture and continue Fiber differentiation 3~4 weeks (being more preferably 3 weeks) generation body embryo, cultivate
Temperature is 25~27 DEG C, dark culturing.
Body embryo is transferred on fresh body embryonal induction culture medium in step (2.2) of the present invention, regulation temperature is 25~27 DEG C,
Body embryo is carried out under dark condition to expand numerous culture, the body embryo expanded after numerous culture is obtained every 1 month subculture once.
Can be carried out according to demand when expanding numerous culture, if desired for body embryo it is many, can many subcultures several times.
Body embryo maturation medium includes following component in step (2.3) of the present invention：MS, sucrose and agar powder, wherein 1L MS
Middle addition has 20~30g of sucrose, 7.5~8.5g of agar powder.
It is preferred that, body embryo maturation medium includes following component in step (2.3) of the present invention：MS, sucrose and agar powder, its
Being added in middle 1L MS has sucrose 25g, agar powder 7.5g.
Wherein the effect of body embryo maturation medium is that body embryo is induced into band green cotyledon type body embryo.MS provides for tissue growth
The nutritional ingredients such as basic N P and K calcium, molysite, trace element and organic matter.Sucrose provides carbon source for the growth of tissue.Agar
Powder is to play a part of to solidify culture medium.
The body embryo expanded after numerous culture is transferred in body embryo maturation medium in step (2.3) of the present invention and cultivated, culture temperature
Spend for 25~27 DEG C, light application time is 15~17h/ days, intensity of illumination is 3000~4000lx, cultivates 7~15d, promotes body embryo
Maturation is green cotyledon type embryo.
It is preferred that, the body embryo expanded after numerous culture is transferred in body embryo maturation medium in step (2.3) of the present invention and cultivated,
Cultivation temperature be 25~27 DEG C, light application time be 16h/ days, intensity of illumination is 4000lx, and light application time is 10d, promotion body embryo into
Ripe is green cotyledon type embryo.
Body embryo germination medium includes following components in step (2.4) of the present invention：MS, 6-BA, sucrose and agar powder, its
0.7~0.9mg of 6-BA, 20~30g of sucrose, 7.5~8.5g of agar powder are added in middle 1L MS.
It is preferred that, 1L body embryo germination mediums include following components in step (2.4) of the present invention：MS, 6-BA, sucrose and
6-BA 0.8mg, sucrose 25g, agar powder 7.5g are added in agar powder, wherein 1L MS.
The effect of body embryo germination medium is induction cotyledon type embryo germination regeneration plant in the present invention.Wherein MS gives birth to for tissue
It is long that the nutritional ingredients such as basic N P and K calcium, molysite, trace element and organic matter are provided.6-BA is the cell point for promoting bud to sprout
Element is split, sucrose provides carbon source for the growth of tissue.Agar powder is to play a part of to solidify culture medium.
Green cotyledon type embryo is transferred in body embryo germination medium in step (2.4) of the present invention and cultivated, cultivation temperature is
25~27 DEG C, light application time is 15~17h/ days, and intensity of illumination is 3000~4000lx, cultivates 25~35d, promotes green cotyledon
Type embryo germination, and then seedling is regenerated, obtain Manihot Esculenta.
It is preferred that, green cotyledon type embryo is transferred in body embryo germination medium in step (2.4) of the present invention and cultivated, culture
Temperature is 25~27 DEG C, and light application time is 16h/ days, and intensity of illumination is 4000lx, cultivates 30d, promotes green cotyledon type embryo to sprout
Hair, and then seedling is regenerated, obtain Manihot Esculenta.
Compared with prior art, the invention has the advantages that：The culture side of cassava fragility embryo callus of the present invention
Method embryonic callus induction generation time of enbrittling is short, the high advantage of generation rate, and cassava is preferably south China 8 and south China
No. 6 are waited conventional cultivation kind.
Brief description of the drawings
Fig. 1 obtains the picture of each tissue, wherein A in the cultural method for cassava fragility embryo callus in embodiment 1
Body embryo block；B：Nascent fragility embryo callus；C：Fragility embryo callus；D：Fragility embryonic callus induction produces body
Embryo；E：Body embryo maturation is cotyledon type embryo；F：Cotyledon type embryo germination is into plant.
Embodiment
Below in conjunction with the accompanying drawings and embodiment, the present invention is described further：
Embodiment 1
The cultural method for the cassava fragility embryo callus that the present embodiment is provided, comprises the following steps：
(1), fragility embryonic callus induction
(1.1) the cassava body embryo block of purifying is placed in the triangular flask equipped with 300mmol/L mannitol, uses breathable sealing film
Sealing；Above-mentioned triangular flask is put into vavuum pump, switch is opened, intake valve is closed, starts meter when air pressure drops to 0.08Mpa
When, after 15min, take out；In the superclean bench sterilized, above-mentioned treated body embryo block is taken out, inhaled with sterilizing filter paper
After solid carbon dioxide point, dark culturing 20d on FECIM is placed on, induction produces nascent fragility embryo callus；
(1.2) under stereomicroscope, nascent fragility embryo callus is transferred on fresh FECIM and breeds expansion
Culture, 2 weeks subcultures once obtain a large amount of fragility embryo callus, extracted available for transgenosis, protoplast etc..
(2) fragility embryo callus regenerates
(2.1) fragility embryo callus is collected into liquid SEIM (being exactly that SEIM is not added with agar powder), concussion and cultivate
1h, removes the soft non-embryonal connective tissue being more than, then outwells liquid SEIM culture mediums, fragility embryo callus is put into SEIM
On culture medium, 25~27 DEG C, dark culturing induction in about 3 weeks produces body embryo；
(2.2) body embryo is transferred on fresh SEIM culture mediums, 25~27 DEG C, dark culturing, body embryo can be carried out to expand numerous
Culture, can be every 1 month subculture once；
(2.3) body embryo is transferred on SEMM, 25~27 DEG C, daily, 3000~4000lx illumination, about 10d promote 16h/
Body embryo maturation is green cotyledon type embryo；
(2.4) green cotyledon type embryo is transferred on SEGM, 25~27 DEG C, 16h/ is daily, 3000~4000lx illumination, about
1 month, promote green cotyledon type embryo germination, and then regenerate seedling.
In the cultural method of above-mentioned cassava fragility embryo callus：
Fragility embryonic callus induction culture medium (Friable embryogenic callus induction
Medium, FECIM) include GD (Gresshof and Doy Basal Medium), 2,4-D, sucrose and agar powder, wherein
1LGD includes 10mg 2,4-D, sucrose 15g, agar powder 10g.
Body embryo inducing culture (somatic embryo induction medium, SEIM) includes NMS, 2,4-D, sugarcane
Sugar and agar powder, it is (Murashige and that 2,4-D 8mg, sucrose 30g, agar powder 8g, NMS are added in wherein 1L NMS
Skoog) MS culture mediums, wherein CaCl2Working concentration is 15mmol/L.
Body embryo maturation medium (somatic embryo maturation medium, SEMM) includes MS, sucrose and fine jade
Cosmetics, wherein 1LMS include sucrose 25g, agar powder 7.5g.
Body embryo germination medium (somatic embryo germination medium, SEGM) includes MS, 6-BA, sugarcane
Sugar and agar powder, wherein 1LMS include 6-BA 0.9mg, sucrose 25g, agar powder 7.5g.
PH to 6.0, autoclave sterilization 20min are adjusted before above-mentioned culture medium, sterilizing.
With it, the initial inductivity of fragility embryo callus is up to more than 90%, and the fragility embryo obtained is cured
Injured tissue can be induced to form body embryo again, and sprout regeneration plant (as shown in fig. 1).
Embodiment 2
The cultural method for the cassava fragility embryo callus that the present embodiment is provided, comprises the following steps：
(1), fragility embryonic callus induction
(1.1) the cassava body embryo block of purifying is placed in the triangular flask equipped with 300mmol/L mannitol, uses breathable sealing film
Sealing；
Above-mentioned triangular flask is put into vavuum pump, switch is opened, intake valve is closed, starts when air pressure drops to 0.08Mpa
After timing, 20min, take out；
In the superclean bench sterilized, above-mentioned treated body embryo block is taken out, after sterilizing filter paper suck dry moisture,
Dark culturing 25d on FECIM is placed on, induction produces nascent fragility embryo callus；
(1.2) under stereomicroscope, nascent fragility embryo callus is transferred on fresh FECIM and breeds expansion
Culture, 3 weeks subcultures once obtain a large amount of fragility embryo callus, extracted available for transgenosis, protoplast etc..
(2) fragility embryo callus regenerates
(2.1) fragility embryo callus is collected into liquid SEIM (being exactly that SEIM is not added with agar powder), concussion and cultivate
1h, removes the soft non-embryonal connective tissue being more than, then outwells liquid SEIM culture mediums, fragility embryo callus is put into SEIM
On culture medium, induction in about 3 weeks produces body embryo, 25~27 DEG C, dark culturing；
(2.2) body embryo is transferred on fresh SEIM culture mediums, 25~27 DEG C, dark culturing, body embryo can be carried out to expand numerous
Culture, can be every 1 month subculture once；
(2.3) body embryo is transferred on SEMM, 25~27 DEG C, 16h, 3000~4000lx illumination, cultivates about 7d, promote body
Embryo maturation is green cotyledon type embryo；
(2.4) green cotyledon type embryo is transferred on SEGM, 25~27 DEG C, 16h, 3000~4000lx illumination, about 35d,
Promote green cotyledon type embryo germination, and then regenerate seedling.
In the cultural method of above-mentioned cassava fragility embryo callus：
Fragility embryonic callus induction culture medium (Friable embryogenic callus induction
Medium, FECIM) include GD (Gresshof and Doy Basal Medium), 2,4-D, sucrose and agar powder, wherein
1LGD includes 8mg 2,4-D, sucrose 18g, agar powder 12.5g.
Body embryo inducing culture (somatic embryo induction medium, SEIM) includes NMS, 2,4-D, sugarcane
Sugar and agar powder, it is MS (Murashige that 2,4-D 10mg, sucrose 25g, agar powder 7.5g, NMS are added in wherein 1L NMS
And Skoog) culture medium, wherein CaCl2Working concentration is 15mmol/L.
Body embryo maturation medium (somatic embryo maturation medium, SEMM) includes MS, sucrose and fine jade
Cosmetics, wherein 1L MS include sucrose 30g, agar powder 8g.
Body embryo germination medium (somatic embryo germination medium, SEGM) includes MS, 6-BA, sugarcane
Sugar and agar powder, wherein 1L MS include 6-BA 0.8mg, sucrose 25g, agar powder 8g.
PH to 6.1, autoclave sterilization 20min are adjusted before above-mentioned culture medium, sterilizing.
With it, the initial inductivity of fragility embryo callus is up to more than 90%, and the fragility embryo obtained is cured
Injured tissue can be induced to form body embryo again, and sprout regeneration plant.
Embodiment 3
The cultural method for the cassava fragility embryo callus that the present embodiment is provided, comprises the following steps：
(1), fragility embryonic callus induction
(1) the cassava body embryo block of south China 8 of purifying is placed in the triangular flask equipped with 250mmol/L mannitol, with ventilative
Sealed membrane is sealed；
(2) above-mentioned triangular flask is put into vavuum pump, opens switch, closed intake valve, opened when air pressure drops to 0.08Mpa
Beginning timing.After 20min, take out；
(3) in the superclean bench sterilized, above-mentioned treated body embryo block is taken out, sterilizing filter paper suck dry moisture is used
Afterwards, dark culturing 30d on FECIM is placed on, induction produces nascent fragility embryo callus；
(4) under stereomicroscope, nascent fragility embryo callus is transferred to propagation on fresh FECIM and expands training
Support, 2 weeks subcultures once obtain a large amount of fragility embryo callus, extracted available for transgenosis, protoplast etc..
(2) fragility embryo callus regenerates
(1) fragility embryo callus is collected into liquid SEIM (being exactly that SEIM is not added with agar powder), concussion and cultivate 1h,
The soft non-embryonal connective tissue being more than is removed, liquid SEIM culture mediums are then outwelled, fragility embryo callus is put into SEIM trainings
Support on base, 25~27 DEG C, dark culturing, induction in about 3 weeks produces body embryo；
(2) body embryo is transferred on fresh SEIM culture mediums, 25~27 DEG C, dark culturing, body embryo can be carried out to expand numerous training
Support, can be every 1 month subculture once；
(3) body embryo is transferred on SEMM, 25~27 DEG C, daily, 3000~4000lx illumination, about 15d promote body to 16h/
Embryo maturation is green cotyledon type embryo；
(4) green cotyledon type embryo is transferred on SEGM, 25~27 DEG C, 16h/ is daily, 3000~4000lx illumination, about
25d, promotes green cotyledon type embryo germination, and then regenerate seedling.
In the cultural method of above-mentioned cassava fragility embryo callus：
Fragility embryonic callus induction culture medium (Friable embryogenic callus induction
Medium, FECIM) include GD (Gresshof and Doy Basal Medium), 2,4-D, sucrose and agar powder, wherein
1LGD includes 2,4-D 12mg, sucrose 20g, agar powder 15g.
Body embryo inducing culture (somatic embryo induction medium, SEIM) includes NMS, 2,4-D, sugarcane
Sugar and agar powder, it is (Murashige and that 2,4-D 9mg, sucrose 20g, agar powder 8.5g, NMS are added in wherein 1L NMS
Skoog) MS culture mediums, wherein CaCl2Working concentration is 15mmol/L.
Body embryo maturation medium (somatic embryo maturation medium, SEMM) includes MS, sucrose and fine jade
Cosmetics, wherein 1L MS include sucrose 30g, agar powder 7.5g.
Body embryo germination medium (somatic embryo germination medium, SEGM) includes MS, 6-BA, sugarcane
Sugar and agar powder, wherein 1L MS include 6-BA 0.7mg, sucrose 25g, agar powder 8g.
PH to 6.2, autoclave sterilization 20min are adjusted before above-mentioned culture medium, sterilizing.
With it, the initial inductivity of fragility embryo callus is up to more than 90%, and the fragility embryo obtained is cured
Injured tissue can be induced to form body embryo again, and sprout regeneration plant.
It will be apparent to those skilled in the art that technical scheme that can be as described above and design, make other various
It is corresponding to change and deformation, and all these change and deformation should all belong to the protection domain of the claims in the present invention
Within.
Claims (10)
1. a kind of cultural method of cassava fragility embryo callus, it is characterized in that comprising the following steps：
(1) fragility embryonic callus induction
(1.1) cassava body embryo block is chosen, after pretreatment, dark culturing on fragility embryonic callus induction culture medium is placed in,
Induction produces nascent fragility embryo callus；
(1.2) nascent fragility embryo callus is transferred to breed on fresh fragility embryonic callus induction culture medium and expanded
Big culture, obtains fragility embryo callus；
(2) fragility embryo callus regenerates
(2.1) fragility embryo callus is collected into liquid bulk embryonal induction culture medium, after concussion and cultivate, removes unnecessary pine
Soft non-embryonal connective tissue, then outwells liquid bulk embryonal induction culture medium, then fragility embryo callus is placed in into body embryo Fiber differentiation
Continue Fiber differentiation in base and produce body embryo；
(2.2) body embryo is transferred on fresh body embryonal induction culture medium, body embryo is carried out under dark condition to expand numerous culture, expanded
Body embryo after numerous culture；
(2.3) body embryo after numerous culture will be expanded and be transferred to culture in body embryo maturation medium, it is green cotyledon type to promote body embryo maturation
Embryo；
(2.4) green cotyledon type embryo is transferred in body embryo germination medium and cultivated, promote green cotyledon type embryo germination, Jin Erzai
Seedling is generated, Manihot Esculenta is obtained.
2. the cultural method of cassava fragility embryo callus according to claim 1, it is characterized in that：In step (1.1)
The pretreatment includes cassava body embryo block being placed in the container containing mannitol, is sealed with breathable sealing film, then by container
It is placed in vavuum pump, regulation air pressure starts to take out after timing, 15~20min when dropping to 0.08Mpa, in the workbench sterilized
In, above-mentioned treated cassava body embryo block is taken out, after sterilizing filter paper suck dry moisture, fragility embryo callus is placed on and lures
Dark culturing 20~30 days on culture medium are led, induction produces nascent fragility embryo callus, and the wherein concentration of mannitol is 250
~300mmol/L.
3. the cultural method of cassava fragility embryo callus according to claim 1, it is characterized in that：Step (1.1)~
(1.2) fragility embryonic callus induction culture medium includes following component in：GD, 2,4-D, sucrose and agar powder, wherein 1L GD
8~12mg of middle addition 2,4-D, 15~20g of sucrose, 10~15g of agar powder.
4. the cultural method of cassava fragility embryo callus according to claim 1, it is characterized in that：In step (1.2)
Nascent fragility embryo callus is transferred on fragility embryonic callus induction culture medium propagation and expands culture, 2~3 weeks after
In generation, can once obtain a large amount of fragility embryo callus.
5. the cultural method of cassava fragility embryo callus according to claim 1, it is characterized in that：In step (2.1)
Body embryo inducing culture includes following component：Add in NMS culture mediums, 2,4-D, sucrose and agar powder, wherein 1L NMS culture mediums
Enter to have 8~10mg of 2,4-D, 20~30g of sucrose, 7.5~8.5g of agar powder.
6. the cultural method of cassava fragility embryo callus according to claim 1, it is characterized in that：In step (2.1)
Fragility embryo callus is collected into liquid bulk embryonal induction culture medium, after 1~2h of concussion and cultivate, removed unnecessary soft non-
Embryonal connective tissue, then outwells liquid bulk embryonal induction culture medium, then fragility embryo callus is placed in body embryo inducing culture
Continue Fiber differentiation and produce within 3~4 weeks body embryo, cultivation temperature is 25~27 DEG C, dark culturing；Step transfers body embryo in (2.2)
Onto fresh body embryonal induction culture medium, regulation temperature is 25~27 DEG C, body embryo is carried out under dark condition to expand numerous culture, every 1
Individual month subculture once, obtains the body embryo expanded after numerous culture.
7. the cultural method of cassava fragility embryo callus according to claim 1, it is characterized in that：In step (2.3)
Body embryo maturation medium includes following component：Being added in MS, sucrose and agar powder, wherein 1L MS has 20~30g of sucrose, agar
7.5~8.5g of powder.
8. the cultural method of cassava fragility embryo callus according to claim 1, it is characterized in that：In step (2.3)
The body embryo expanded after numerous culture is transferred in body embryo maturation medium and cultivated, cultivation temperature is 25~27 DEG C, and light application time is 15
~17h/ days, intensity of illumination was 3000~4000lx, cultivates 7~15d, and it is green cotyledon type embryo to promote body embryo maturation.
9. the cultural method of cassava fragility embryo callus according to claim 1, it is characterized in that：In step (2.4)
Body embryo germination medium includes following components：In MS, 6-BA, sucrose and agar powder, wherein 1L MS add 6-BA 0.7~
0.9mg, 20~30g of sucrose, 7.5~8.5g of agar powder.
10. the cultural method of cassava fragility embryo callus according to claim 1, it is characterized in that：In step (2.4)
By green cotyledon type embryo be transferred in body embryo germination medium cultivate, cultivation temperature be 25~27 DEG C, light application time be 15~
17h/ days, intensity of illumination was 3000~4000lx, cultivates 25~35d, promoted green cotyledon type embryo germination, and then regeneration seedling,
Obtain Manihot Esculenta.
Priority Applications (1)
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| CN109566410A (en) * | 2018-06-13 | 2019-04-05 | 中国热带农业科学院热带作物品种资源研究所 | A kind of cultural method of cassava axillary bud somatic embryo in vitro culture detoxic seedling |
| CN107446878B (en) * | 2017-09-07 | 2020-06-30 | 海南大学 | Method for quickly and efficiently obtaining cassava somatic embryos |
| CN114438115A (en) * | 2021-12-23 | 2022-05-06 | 中国热带农业科学院热带生物技术研究所 | CRISPR/Cas9 gene editing vector, construction method and application thereof |
| CN114847164A (en) * | 2022-06-09 | 2022-08-05 | 中国热带农业科学院热带生物技术研究所 | Brittle embryogenic callus of cassava 60 Co-gamma ray radiation mutation breeding method |
| CN117223609A (en) * | 2023-11-01 | 2023-12-15 | 中国热带农业科学院热带作物品种资源研究所 | Method for shortening cassava regeneration plant period |
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