CN107142277A - A kind of efficient long-grained nonglutinous rice genetic transforming method - Google Patents
A kind of efficient long-grained nonglutinous rice genetic transforming method Download PDFInfo
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Abstract
The invention discloses a kind of efficient long-grained nonglutinous rice genetic transforming method, this method includes：Induction, callus preculture, the culture of Agrobacterium and the Agrobacterium-medialed transformation of long-grained nonglutinous rice mature embryo callus, the co-cultivation of Agrobacterium and callus, the screening of resistant calli, differentiation and regrowth are taken root；This method simple and effective, the callus quality obtained is good, it is adapted to conversion, conversion operation simple flow, by being that restorer 2537 is converted to rice variety indica type two-line sterile line 628S and indica type two, discovery can obtain long-grained nonglutinous rice positive transformants plant for 60 days or so, and transformation efficiency is up to more than 35%, conversion ratio is higher, improves the genetic transformation efficiency of long-grained nonglutinous rice.
Description
Technical field
The invention belongs to Plant Tissue Breeding and gene engineering technique field, in particular to a kind of efficient long-grained nonglutinous rice
Genetic transforming method, the present invention is applied to the callus of induction and subculture long-grained nonglutinous rice mature seed, and agriculture bacillus mediated embryo
The genetic transforming method of property callus.
Background technology
Paddy rice (Oryza sativa L.) is one of most important cereal crops of China, is divided into long-grained nonglutinous rice and japonica rice two is big sub-
Kind, its medium rice is the maximum cultivated rice type of China, and cultivated area accounts for the 74% of national rice area.Utilize transgenosis skill
Art is improved to rice varieties, and culture high yield, high-quality, many anti-, extensively suitable new varieties are while ensureing that world food is safe
It is also the developing direction of rice breeding from now on.Therefore, the genetic transformation of the genetic transformation of paddy rice, especially long-grained nonglutinous rice changes in kind
More and more important effect is played in good, gene function detection, creation mutant, and sets up long-grained nonglutinous rice heredity efficiently, stable
Transformation system is the premise of the area researches such as molecular breeding and functional genomics.
In recent years, rice transformation technology achieves important breakthrough, and quick, height is largely established in japonica rice
Effect, stable genetic conversion system.On this basis, by many important characters are for example pest-resistant, disease-resistant, degeneration-resistant, quality-improving,
Developmental regulation, nutrient efficient such as utilize to be transferred in paddy rice at the foreign gene.But for the larger long-grained nonglutinous rice of cultivated area, due to
The difference of genotype causes its Culture characteristics generally poor, and conversion difficulty is significantly greater than japonica rice.Soil is utilized in recent years
Agrobacterium-mediated transformation long-grained nonglutinous rice also achieves some successes, but not yet sets up so far a set of relatively stable as japonica rice
Efficient genetic trasformation system.
There are some researches show agriculture bacillus mediated genetic transformation efficiency and the callus status of recipient genotypes, culture
The factors such as base, mode of infection are relevant.Therefore, by optimizing the parameter in long-grained nonglutinous rice genetic transformation each stage, it is expected to set up efficient long-grained nonglutinous rice
Genetic conversion system.
The content of the invention
It is an object of the invention to provide a kind of efficient long-grained nonglutinous rice genetic transforming method, the callus quality that this method is obtained
It is good, it is adapted to conversion, conversion operation simple flow, conversion ratio is higher, and 2 rice varieties tested all obtain success, conversion
Efficiency is between 35%-40%.
The present invention is achieved through the following technical solutions：
A kind of efficient long-grained nonglutinous rice genetic transforming method, comprises the following steps：
(1) induction of callus
Choose the grain of rice of ripe good, complete long-grained nonglutinous rice seed, first with 75% alcohol disinfecting 3 minutes, then be soaked in
25% (V/V) liquor natrii hypochloritis, is placed in 150rpm, sterilizes 20 minutes on 25 DEG C of shaking table；, will on superclean bench
Seed after above-mentioned sterilization sterile water washing 4-5 times, is then transferred to suck dry moisture on aseptic filter paper, then by appropriate seed
NB culture mediums are inoculated in, illumination cultivation 8-10 days, evoked callus at 28-30 DEG C；
(2) preculture of callus
Select that compact structure, color be vivid, granular callus is transferred to pre- training in step (1) described callus
Support in base, be used within 3 days convert in illumination cultivation at 28-30 DEG C；
(3) culture of Agrobacterium and Agrobacterium-medialed transformation
Plant expression vector containing target gene is transferred to Agrobacterium EHA105 by Electroporation conversion, positive bacteria is chosen
Strain lines YEB resistant panels, and 28 DEG C of light cultures 3 days, then the Agrobacterium concussion for scraping grain of rice size with transfer needle are suspended in dress
In the 150ml triangular flasks for having 70ml During Agrobacterium liquid, 150rpm is placed in, 10 minutes are shaken on 28 DEG C of shaking table, then will step
Suddenly (2) described 3 days Indica Rice Callus of illumination cultivation is soaked in above-mentioned During Agrobacterium liquid, is shaken in 150rpm, 28 DEG C
Shaken 10 minutes on bed；
(4) co-cultivation of Agrobacterium and callus
Indica Rice Callus after step (3) described dip-dye is placed on aseptic filter paper and dried up, then by the callus group
Knit and be transferred on co-cultivation base, co-culture advance one aseptic filter paper of pad of base flat board, drenched with 1ml During Agrobacterium liquid, and
Remove bubble, 28 DEG C of light cultures 3 days；
(5) screening of resistant calli
By step (4) it is described co-culture 3 days after callus be placed in 250ml triangular flasks, with sterile water washing 4-5
It is secondary, untill cleaning solution is as clear as crystal；Then the carbenicillin solution washing for adding the 500mg/L of filtration sterilization is described more
Injured tissue, room temperature is placed in shaking table 150rpm concussion washing 15min, then the callus is placed on aseptic filter paper blots water
Point, it is inoculated on the screening and culturing medium containing corresponding selective agent and screens, the condition of the screening and culturing is 28 DEG C, illumination cultivation
20-22 days, middle subculture once, until growing resistant calli；
(6) differentiation of resistant calli
The resistant calli obtained after step (5) described screening is transferred on the differential medium containing selective agent, in
28 DEG C, illumination cultivation 18-23 days, middle subculture once, makes its Shoot regeneration；
(7) regrowth is taken root
The seedling grown on step (6) described differential medium is transferred in the root media containing selective agent, 30 DEG C,
Illumination cultivation 10-12 days, obtains regrowth.
NB nutrient media componentses described in step (1) are：N6Inorganic salts and organic matter+inositol 0.1g/L+ glutamine
0.5g/L+ acid hydrolyzed casein 0.5g/L+ proline 2.878g/L+ sucrose 30.0g/L+ agar powders 8.0g/L+2,4-D
2.0mg/L, pH are 5.8.
Pre-culture medium component described in step (2) is：N6Inorganic salts and organic matter+inositol 0.1g/L+ glutamine
0.5g/L+ acid hydrolyzed casein 0.5g/L+ proline 2.878g/L+ sucrose 30.0g/L+ agar powders 8.0g/L+2,4-D
2.0mg/L+ acetosyringones 0.1mM, pH are 5.8.
During Agrobacterium liquid component described in step (3) is：AA inorganic salts and organic matter+inositol 0.1g/L+ sour water solutions
Casein 0.5g/L+ sucrose 68.5g/L+ glucose 36.0g/L+ glutamine 0.9g/L+ asparagine 0.3g/L+ arginine
0.176g/L, pH are 5.2；Use preceding addition acetosyringone 0.1mM.
Co-cultivation base component described in step (4) is：N6Inorganic salts and organic matter+inositol 0.1g/L+ sour water solution junket eggs
White 0.3g/L+ sucrose 30.0g/L+ glucose 10.0g/L+ agar powders 8.0g/L+2,4-D2.0mg/L, pH are 5.2, are added after sterilizing
Enter acetosyringone 0.1mM.
Screening and culturing medium component described in step (5) is：N6Inorganic salts and organic matter+inositol 0.1g/L+ sour water solution junket
Albumen 0.5g/L+ proline 2.878g/L+ glutamine 0.3g/L+ sucrose 30.0g/L+ phytolectins 4.0g/L+2,4-D
2.0mg/L, pH are 5.8；Carbenicillin 400mg/L+ hygromycin 30mg/L or Basta 4mg/L are added after sterilizing.
Differential medium component described in step (6) is：MS inorganic salts and organic matter+inositol 0.1g/L+ sour water solution junket
Albumen 2.0g/L+ proline 0.3g/L+ sucrose 30.0g/L+ sorbierite 30g/L+ phytolectin 4.0g/L+NAA 0.02mg/L
+ Kinetin2mg/L, pH are 5.8；Carbenicillin 400mg/L+ hygromycin 30mg/L or Basta4mg/L are added after sterilizing.
Root media component described in step (7) is：MS inorganic salts and organic matter+inositol 0.1g/L+ sour water solution junket
Albumen 2.0g/L+ proline 0.3g/L+ sucrose 30.0g/L+ phytolectins 4.0g/L, pH are 5.8；Carboxylic benzyl is added after sterilizing blue or green
Mycin 400mg/L.
The advantage of the invention is that：
The present invention is that restorer 2537 carries out agriculture to 2 long-grained nonglutinous rice representativeness kind indica type two-line sterile line 628S and indica type two
The conversion of bacillus EHA105 mediations, transformation efficiency Gao Jun is more than 35%；The callus status of this method induction is good, is adapted to turn
Change, whole conversion operation process cycle is shorter.
Brief description of the drawings
Below in conjunction with the accompanying drawings and embodiment the present invention will be further described.
Fig. 1 is restorer 2537 (a) and the ripe embryonal induction of indica type two-line sterile line 628S (b) in the present invention indica type two
Callus；
Fig. 2 is that restorer 2537 (a) and indica type two-line sterile line 628S (b) callus are trained in advance in the present invention indica type two
Support；
Fig. 3 in the present invention indica type two be restorer 2537 (a) and indica type two-line sterile line 628S (b) screen 10 days more
Injured tissue；
Fig. 4 in the present invention indica type two be restorer 2537 (a) and indica type two-line sterile line 628S (b) break up 10 days more
Injured tissue；
Indica type two be to be produced after restorer 2537 (a) is broken up with indica type two-line sterile line 628S (b) in Fig. 5 present invention
Seedling carries out culture of rootage；
Fig. 6 is restorer 2537 (a) and indica type two-line sterile line 628S (b) part T in the present invention indica type two0For plant
The GUS dyeing identifications of blade；
Fig. 7 is restorer 2537 (a) and indica type two-line sterile line 628S (b) part T in the present invention indica type two0For plant
The PCR detection electrophoretograms of hygromycin gene.
Embodiment
Following examples are used to illustrate the present invention, but are not limited to the scope of the present invention.
Embodiment 1
(1) induction of callus
The grain of rice that ripe good, complete rice variety indica type two is restorer 2537 is chosen, is first disappeared with 75% alcohol
Poison 3 minutes, then is soaked in 25% (V/V) liquor natrii hypochloritis, is placed in 150rpm, sterilizes 20 minutes on 25 DEG C of shaking table；Disappear
Then seed after poison uses aseptic filter paper suck dry moisture, then seed is inoculated in into NB in superclean bench sterile water washing 5 times
Culture medium；Illumination cultivation 8 days at 28 DEG C -30 DEG C, evoked callus (Fig. 1 a), wherein, NB nutrient media componentses are：N6It is inorganic
Salt and organic matter+inositol 0.1g/L+ glutamine 0.5g/L+ acid hydrolyzed casein 0.5g/L+ proline 2.878g/L+ sucrose
30.0g/L+ agar powders 8.0g/L+2,4-D 2.0mg/L, pH are 5.8.
(2) preculture of callus
Select that compact structure, color be vivid, granular callus is transferred in pre-culture medium, in light at 28 DEG C -30 DEG C
According to culture 3 days, for converting (Fig. 2 a), wherein, pre-culture medium component is：N6Inorganic salts and organic matter+inositol 0.1g/L+ paddy ammonia
Acid amides 0.5g/L+ acid hydrolyzed casein 0.5g/L+ proline 2.878g/L+ sucrose 30.0g/L+ agar powders 8.0g/L+2,4-D
2.0mg/L+ acetosyringones 0.1mM, pH are 5.8.
(3) culture of Agrobacterium and Agrobacterium-medialed transformation
Plant expression vector containing target gene is transferred to EHA105 by Electroporation conversion, by the positive after conversion
EHA105 agrobacterium strains line YEB resistant panels, 28 DEG C of light cultures 3 days, then scrape with transfer needle the agriculture bar of grain of rice size
Bacterium, concussion is suspended in the 150ml triangular flasks equipped with 70ml During Agrobacterium liquid, is placed in 150rpm, is shaken 10 on 28 DEG C of shaking table
Minute；Meanwhile, the callus after preculture 3 days is soaked in During Agrobacterium liquid, 10 are shaken on 150rpm, 28 DEG C of shaking table
Minute；Wherein, During Agrobacterium liquid component is：AA inorganic salts and organic matter+inositol 0.1g/L+ acid hydrolyzed caseins 0.5g/L+
Sucrose 68.5g/L+ glucose 36.0g/L+ glutamine 0.9g/L+ asparagine 0.3g/L+ arginine 0.176g/L, pH are
5.2；Use preceding addition acetosyringone 0.1mM.
(4) co-cultivation of Agrobacterium and callus
Callus after dip-dye is placed in suck dry moisture on aseptic filter paper standby.Co-culture one, pad on base flat board sterile
Filter paper, is then drenched with 1ml During Agrobacterium liquid, and removes bubble, then shifts the callus of advance suck dry moisture
To co-culturing in base, 28 DEG C of light cultures 3 days；Wherein, co-culturing base component is：N6Inorganic salts and organic matter+inositol 0.1g/L+ acid
Caseinhydrolysate 0.3g/L+ sucrose 30.0g/L+ glucose 10.0g/L+ agar powders 8.0g/L+2,4-D 2.0mg/L, pH are
5.2, acetosyringone 0.1mM is added after sterilizing.
(5) screening of resistant calli
Callus after co-culturing 3 days is placed in 250ml triangular flasks, with sterile water washing 4 times, until cleaning solution is clear
It is clear it is transparent untill.The 500mg/L of filtration sterilization carbenicillin solution washing callus is added, room temperature is placed in shaking table
150rpm concussion washing 15min, then callus is placed in suck dry moisture on aseptic filter paper, it is inoculated in containing corresponding selective agent
Screened on screening and culturing medium；The condition of screening and culturing is 28 DEG C, and illumination cultivation 20 days, every 10 days subcultures are once, anti-until growing
Property callus (Fig. 3 a), wherein, screening and culturing medium component is：N6Inorganic salts and organic matter+inositol 0.1g/L+ sour water solution junket eggs
White 0.5g/L+ proline 2.878g/L+ glutamine 0.3g/L+ sucrose 30.0g/L+ phytolectins 4.0g/L+2,4-D
2.0mg/L, pH are 5.8；Carbenicillin 400mg/L+ hygromycin 30mg/L or Basta 4mg/L are added after sterilizing.
(6) differentiation of resistant calli
The resistant calli obtained after screening is transferred on the differential medium containing selective agent, in 28 DEG C, illumination training
Support 20 days, every 10 days subcultures once, make its Shoot regeneration (Fig. 4 a), wherein, differential medium component is：MS inorganic salts and organic
Thing+inositol 0.1g/L+ acid hydrolyzed casein 2.0g/L+ proline 0.3g/L+ sucrose 30.0g/L+ sorbierite 30g/L+ plants coagulate
It is 5.8 to collect element 4.0g/L+NAA 0.02mg/L+Kinetin2mg/L, pH；Carbenicillin 400mg/L+ tides are added after sterilizing mould
Plain 30mg/L or Basta4mg/L.
(7) regrowth is taken root
The seedling grown in differentiation culture is transferred in the root media containing selective agent, 30 DEG C, illumination cultivation 10 days,
Obtain regrowth；Wherein, root media component is：MS inorganic salts and organic matter+inositol 0.1g/L+ acid hydrolyzed caseins
2.0g/L+ proline 0.3g/L+ sucrose 30.0g/L+ phytolectins 4.0g/L, pH are 5.8；Carbenicillin is added after sterilizing
400mg/L。
Embodiment 2
(1) choose ripe good, the complete rice variety 628S grain of rice, first with 75% alcohol disinfecting 3 minutes, then soak
Steep in 25% (V/V) liquor natrii hypochloritis, be placed in 150rpm, sterilize 20 minutes on 25 DEG C of shaking table.Seed after sterilization
In superclean bench sterile water washing 5 times, then with suck dry moisture on aseptic filter paper, then seed is inoculated in NB culture mediums.
Illumination cultivation 10 days, evoked callus (Fig. 1 b) at 28 DEG C~30 DEG C；NB nutrient media componentses are：N6Inorganic salts and organic matter
+ inositol 0.1g/L+ glutamine 0.5g/L+ acid hydrolyzed casein 0.5g/L+ proline 2.878g/L+ sucrose 30.0g/L+ agar
Powder 8.0g/L+2,4-D 2.0mg/L, pH are 5.8..
(2) preculture of callus
Select that compact structure, color be vivid, granular callus is transferred in pre-culture medium, in light at 28 DEG C -30 DEG C
According to culture 3 days, for converting (Fig. 2 b)；Pre-culture medium component is：N6Inorganic salts and organic matter+inositol 0.1g/L+ glutamine
0.5g/L+ acid hydrolyzed casein 0.5g/L+ proline 2.878g/L+ sucrose 30.0g/L+ agar powders 8.0g/L+2,4-D
2.0mg/L+ acetosyringones 0.1mM, pH are 5.8.
(3) culture of Agrobacterium and Agrobacterium-medialed transformation
Plant expression vector containing target gene is transferred to EHA105 by Electroporation conversion.By the positive after conversion
EHA105 bacterial strains line YEB resistant panels, 28 DEG C of light cultures 3 days, then scrape with transfer needle the Agrobacterium of grain of rice size, concussion
It is suspended in the 150ml triangular flasks equipped with 70ml During Agrobacterium liquid, is placed in 150rpm, shakes 10 minutes on 28 DEG C of shaking table；Together
When, the Indica Rice Callus after preculture 3 days is soaked in During Agrobacterium liquid, 10 points are shaken on 150rpm, 28 DEG C of shaking table
Clock；During Agrobacterium liquid component is：AA inorganic salts and organic matter+inositol 0.1g/L+ acid hydrolyzed casein 0.5g/L+ sucrose
68.5g/L+ glucose 36.0g/L+ glutamine 0.9g/L+ asparagine 0.3g/L+ arginine 0.176g/L, pH are 5.2；Make
With preceding addition acetosyringone 0.1mM.
(4) co-cultivation of Agrobacterium and callus
Callus after dip-dye is placed in suck dry moisture on aseptic filter paper standby.Co-culture one, pad on base flat board sterile
Filter paper, is then drenched with 1ml During Agrobacterium liquid, and removes bubble, then shifts the callus of advance suck dry moisture
To co-culturing in base, 28 DEG C of light cultures 3 days；Co-culturing base component is：N6Inorganic salts and organic matter+inositol 0.1g/L+ sour water solutions
Casein 0.3g/L+ sucrose 30.0g/L+ glucose 10.0g/L+ agar powders 8.0g/L+2,4-D 2.0mg/L, pH are 5.2, are gone out
Acetosyringone 0.1mM is added after bacterium.
(5) screening of resistant calli
Callus after co-culturing 3 days is placed in 250ml triangular flasks, with sterile water washing 4 times, until cleaning solution is clear
It is clear it is transparent untill.The 500mg/L of filtration sterilization carbenicillin solution washing callus is added, room temperature is placed in shaking table
150rpm concussion washing 15min, then callus is placed in suck dry moisture on aseptic filter paper, it is inoculated in containing corresponding selective agent
Screened on screening and culturing medium.The condition of screening and culturing is 28 DEG C, and illumination cultivation 22 days, every 11 days subcultures are once, anti-until growing
Property callus (Fig. 3 b)；Screening and culturing medium component is：N6Inorganic salts and organic matter+inositol 0.1g/L+ acid hydrolyzed caseins
0.5g/L+ proline 2.878g/L+ glutamine 0.3g/L+ sucrose 30.0g/L+ phytolectins 4.0g/L+2,4-D
2.0mg/L, pH are 5.8；Carbenicillin 400mg/L+ hygromycin 30mg/L or Basta 4mg/L are added after sterilizing.
(6) differentiation of resistant calli
The resistant calli obtained after screening is transferred on the differential medium containing selective agent, in 28 DEG C, illumination training
Support 18 days, every 9 days subcultures once, make its Shoot regeneration (Fig. 4 b)；Differential medium component is：MS inorganic salts and organic matter+flesh
Alcohol 0.1g/L+ acid hydrolyzed casein 2.0g/L+ proline 0.3g/L+ sucrose 30.0g/L+ sorbierite 30g/L+ phytolectins
4.0g/L+NAA0.02mg/L+Kinetin2mg/L, pH are 5.8；Carbenicillin 400mg/L+ hygromycin is added after sterilizing
30mg/L or Basta4mg/L.
(7) regrowth is taken root
The seedling grown in differentiation culture is transferred in the root media containing selective agent, 30 DEG C, illumination cultivation 12 days,
Obtain regrowth (Fig. 5 b)；Root media component is：MS inorganic salts and organic matter+inositol 0.1g/L+ acid hydrolyzed caseins
2.0g/L+ proline 0.3g/L+ sucrose 30.0g/L+ phytolectins 4.0g/L, pH are 5.8；Carbenicillin is added after sterilizing
400mg/L。
In embodiment 1, the present invention indica type to rice variety two is that the callus of restorer 2537 is converted, by 63
Transformed calli carries out hygromycin resistance screening, and carries out GUS dyeing (Fig. 6 a) and PCR detection (figures for plant leaf to T0
7a), 24 positive transgenic plant are finally given, its transformation efficiency has reached 38.1%.
In example 2, the present invention is converted to indica type two-line sterile line 628S callus, and callus is converted by 54
Tissue carries out hygromycin resistance screening, and carries out GUS dyeing (Fig. 6 b) and PCR detections (Fig. 7 b) for plant leaf to T0, finally
20 positive transgenic plant are obtained, its transformation efficiency has reached 37.04%.
Although above with general explanation and specific embodiment, the present invention is described in detail, at this
On the basis of invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Therefore,
These modifications or improvements, belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (8)
1. a kind of efficient long-grained nonglutinous rice genetic transforming method, it is characterised in that comprise the following steps：
Step 1：The induction of callus
Choose the grain of rice of ripe good, complete long-grained nonglutinous rice seed, first with 75% alcohol disinfecting 3 minutes, then be soaked in 25% (V/
V liquor natrii hypochloritis), is placed in 150rpm, sterilizes 20 minutes on 25 DEG C of shaking table；On superclean bench, disappear above-mentioned
Seed after poison sterile water washing 4-5 times, is then transferred to suck dry moisture on aseptic filter paper, then appropriate seed is inoculated in
NB culture mediums, illumination cultivation 8-10 days, evoked callus at 28-30 DEG C；
Step 2：The preculture of callus
Select that compact structure, color be vivid, granular callus is transferred to pre-culture medium in callus described in step 1
In, it is used within 3 days convert in illumination cultivation at 28-30 DEG C；
Step 3：The culture of Agrobacterium and Agrobacterium-medialed transformation
Plant expression vector containing target gene is transferred to agrobacterium strains EHA105 by Electroporation conversion, positive bacteria is chosen
Strain lines YEB resistant panels, and 28 DEG C of light cultures 3 days, then the Agrobacterium concussion for scraping grain of rice size with transfer needle are suspended in dress
In the 150ml triangular flasks for having 70ml During Agrobacterium liquid, 150rpm is placed in, 10 minutes are shaken on 28 DEG C of shaking table, then will step
The illumination cultivation Indica Rice Callus of 3 days is soaked in above-mentioned During Agrobacterium liquid described in rapid 2, in 150rpm, 28 DEG C of shaking table
On shake 10 minutes；
Step 4：Agrobacterium and the co-cultivation of callus
Indica Rice Callus after being contaminated described in step 3 is placed on aseptic filter paper and dried up, then the callus is shifted
To co-culturing on base, advance one aseptic filter paper of pad of base flat board is co-cultured, is drenched with 1ml During Agrobacterium liquid, and except degassing
Bubble, 28 DEG C of light cultures 3 days；
Step 5：The screening of resistant calli
Callus after being co-cultured 3 days described in step 4 is placed in 250ml triangular flasks, with sterile water washing 4-5 time, up to
Untill cleaning solution is as clear as crystal；Then the carbenicillin solution for adding the 500mg/L of filtration sterilization washs the callus,
Room temperature is placed in shaking table 150rpm concussion washing 15min, then the callus is placed in into suck dry moisture on aseptic filter paper, is inoculated in
Screened on screening and culturing medium containing corresponding selective agent, the condition of the screening and culturing is 28 DEG C, illumination cultivation 20-22 days, in
Between subculture once, until growing resistant calli；
Step 6：The differentiation of resistant calli
The resistant calli obtained after being screened described in step 5 is transferred on the differential medium containing selective agent, in 28 DEG C, light
According to culture 18-23 days, middle subculture once, made its Shoot regeneration；
Step 7：Regrowth is taken root
The seedling grown described in step 6 on differential medium is transferred in the root media containing selective agent, 30 DEG C, illumination training
Support 10-12 days, obtain regrowth.
2. a kind of efficient long-grained nonglutinous rice genetic transforming method according to claim 1, it is characterised in that the NB described in step 1
Nutrient media components is：N6Inorganic salts and organic matter+inositol 0.1g/L+ glutamine 0.5g/L+ acid hydrolyzed casein 0.5g/L+ dried meat
Propylhomoserin 2.878g/L+ sucrose 30.0g/L+ agar powders 8.0g/L+2,4-D 2.0mg/L, pH are 5.8.
3. a kind of efficient long-grained nonglutinous rice genetic transforming method according to claim 1, it is characterised in that pre- described in step 2
Nutrient media components is：N6Inorganic salts and organic matter+inositol 0.1g/L+ glutamine 0.5g/L+ acid hydrolyzed casein 0.5g/L+ dried meat
Propylhomoserin 2.878g/L+ sucrose 30.0g/L+ agar powders 8.0g/L+2,4-D 2.0mg/L+ acetosyringones 0.1mM, pH are 5.8.
4. a kind of efficient long-grained nonglutinous rice genetic transforming method according to claim 1, it is characterised in that the agriculture described in step 3
Bacillus dip dyeing liquid for shell component is：AA inorganic salts and organic matter+inositol 0.1g/L+ acid hydrolyzed casein 0.5g/L+ sucrose 68.5g/L+
Glucose 36.0g/L+ glutamine 0.9g/L+ asparagine 0.3g/L+ arginine 0.176g/L, pH are 5.2；Added using preceding
Acetosyringone 0.1mM.
5. a kind of efficient long-grained nonglutinous rice genetic transforming method according to claim 1, it is characterised in that being total to described in step 4
Nutrient media components is：N6Inorganic salts and organic matter+inositol 0.1g/L+ acid hydrolyzed casein 0.3g/L+ sucrose 30.0g/L+ grapes
Sugared 10.0g/L+ agar powders 8.0g/L+2,4-D 2.0mg/L, pH are 5.2, and acetosyringone 0.1mM is added after sterilizing.
6. a kind of efficient long-grained nonglutinous rice genetic transforming method according to claim 1, it is characterised in that the sieve described in step 5
The nutrient media components is selected to be：N6Inorganic salts and organic matter+inositol 0.1g/L+ acid hydrolyzed casein 0.5g/L+ proline 2.878g/L+
Glutamine 0.3g/L+ sucrose 30.0g/L+ phytolectins 4.0g/L+2,4-D 2.0mg/L, pH are 5.8；Added after sterilizing
Carbenicillin 400mg/L+ hygromycin 30mg/L or Basta 4mg/L.
7. a kind of efficient long-grained nonglutinous rice genetic transforming method according to claim 1, it is characterised in that point described in step 6
Changing nutrient media components is：MS inorganic salts and organic matter+inositol 0.1g/L+ acid hydrolyzed casein 2.0g/L+ proline 0.3g/L+ sugarcanes
Sugared 30.0g/L+ sorbierites 30g/L+ phytolectin 4.0g/L+NAA 0.02mg/L+Kinetin2mg/L, pH are 5.8；Sterilizing
Carbenicillin 400mg/L+ hygromycin 30mg/L or Basta4mg/L are added afterwards.
8. a kind of efficient long-grained nonglutinous rice genetic transforming method according to claim 1, it is characterised in that the life described in step 7
Root nutrient media components is：MS inorganic salts and organic matter+inositol 0.1g/L+ acid hydrolyzed casein 2.0g/L+ proline 0.3g/L+ sugarcanes
Sugared 30.0g/L+ phytolectins 4.0g/L, pH are 5.8；Carbenicillin 400mg/L is added after sterilizing.
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| CN113637701A (en) * | 2021-09-10 | 2021-11-12 | 中国农业科学院烟草研究所(中国烟草总公司青州烟草研究所) | Method for establishing wild rice genetic transformation system |
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