CN104988180B - A kind of preparation method of Salix suchowensis resistance seedling - Google Patents
A kind of preparation method of Salix suchowensis resistance seedling Download PDFInfo
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Abstract
The invention discloses a kind of preparation method of Salix suchowensis resistance seedling, including explant and prepare and preculture, infect the preparation of bacterium solution, infect, co-culture, wash bacterium, differentiation screening and culturing is until differentiate visible kalamycin resistance bud, it is transferred on Elongation of adventitious bud screening and culturing medium and continues to cultivate, treat that resistant buds are extended to 1 2cm, each resistant buds are separated independently, moves into screening and culturing medium of taking root and cultivates, complete resistance seedling is can obtain through squamous subculture after a while.This method includes the system preparation method that Salix suchowensis resistance seedling is established out from explant selection, Screening of Media, hormone allotment, antibiotics sensitivity, condition of culture etc., and new way is provided for forest gene functional research.
Description
Technical field
The invention belongs to gene engineering technology field, relates in particular to a kind of preparation method of Salix suchowensis resistance seedling.
Background technology
Salix suchowensis (Salix suchowensis Cheng), Salicaceae (Salicaceae) Salix (Salix), clump of falling leaves
Raw perennial shrub plant, branch are in yellow green or purplish, leaf alternate, lanceolar, and length is hairless more than 10cm.Hua Xian
Leaf opens, and the florescence is March, and fruiting period is in the 4-5 months.The place of production is mainly distributed on the ground such as Basin of Huaihe River, Zhejiang, Jiangsu, Shandong, Henan
There is cultivation.Salix suchowensis individual is small, and the generation cycle is short, can bloom within general 1 year, natural variation is enriched.These features cause dustpan
Willow is the ideal material for carrying out forest tree genetic research more suitable for carrying out extensive field trial.
Genetic transfoumation is the important means for studying gene function, but up to the present, people are to xylophyta base
Because the correlative study of function is also very limited, the Study on Genetic Transformation on willow also rests on the desk study rank of regenerating system
Section, does not obtain breakthrough achievement in research.Therefore, Salix suchowensis tissue culture regeneration is studied, and establishes stable genetic transformation
System, the Study on Genetic Transformation for carrying out Salix suchowensis or even Salicaceous Plants on a large scale from now on, promotes forest functional genome
Research is of great significance.
The content of the invention
Goal of the invention：For the deficiencies in the prior art, the object of the present invention is to provide a kind of Salix suchowensis resistance seedling
Preparation method, provide a new way for forest tree gene functional research and genetic improvement.
Technical solution：In order to realize foregoing invention purpose, the technical solution adopted by the present invention is：
A kind of preparation method of Salix suchowensis resistance seedling, comprises the following steps：
1) Salix suchowensis tissue-cultured seedling is placed in successive propagation on minimal medium；
2) the tissue-cultured seedling stem of subculture is chosen, is cut into the stem section of 1-2cm long, the blade of surrounding is cut off, is inoculated with as explant
In preculture 2-3d on differential medium；Differential medium is WP culture mediums, additional cellular mitogen 6-BA 0.1-1.0mg/L,
Basic element of cell division TDZ 0.005mg/L, auxin IBA 0.01-0.005mg/L, sucrose 30g/L, crystal agar 2g/L；
3) colony inoculation of picking Agrobacterium after shaken cultivation, takes portion into the LB fluid nutrient mediums containing kanamycins
Divide and be forwarded in LB fluid nutrient mediums, treat that thalline enters exponential phase of growth, thalline is obtained by centrifugation, takes WP liquid to support base culture
Bacterium solution is infected in base suspension thalline, acquisition；
4) stem section after the preculture of step 2) is immersed to the bacterium solution that infects of step 3), vibration, after soaking 10-20min, is removed
Unnecessary bacterium solution is gone, puts back on differential medium and is co-cultured；
5) Agrobacterium is removed to the stem section washing after co-cultivation, is transferred on differentiation screening and culturing medium and cultivates, regularly replace
Break up screening and culturing medium, until differentiating visible kalamycin resistance bud；
6) kalamycin resistance bud is even transferred on Elongation of adventitious bud screening and culturing medium and continues to cultivate；
7) treat that resistant buds are extended to 1-2cm, each resistant buds separated independently, moves into screening and culturing medium of taking root and cultivates,
Complete resistance seedling is can obtain through squamous subculture after a while.
In step 1), the minimal medium is 1/2WP, additional saccharose 25-30g/L, crystal agar 2g/L, pH
5.8,121 ± 1 DEG C, sterilize 18min.
In step 2), the preculture condition is 25 ± 2 DEG C, illumination 3000-4000lux of cultivation temperature, light application time
16h/d。
In step 2), the differential medium is WP culture mediums, additional cellular mitogen 6-BA 0.5mg/L, cell division
Plain TDZ 0.005mg/L, auxin IBA 0.01mg/L, sucrose 30g/L, crystal agar 2g/L.
In step 3), the concentration of kanamycins is 40mg/L in the LB fluid nutrient mediums.
In step 3), the OD of the WP fluid nutrient mediums suspension thalline600It is worth for 0.6.
In step 5), the differentiation screening and culturing medium is in differential medium, and it is 200mg/ to add cephalosporin concentration
L, kanamycins concentration are 40mg/L.
In step 6), the Elongation of adventitious bud screening and culturing medium is WP, additional cellular mitogen 6-BA 0.5mg/L, is grown
Plain IBA 0.01mg/L, cephalosporin 200mg/L, kanamycins 40mg/L.
In step 7), the screening and culturing medium of taking root is 1/2WP, and cephalosporin concentration is 100mg/L, kanamycins it is dense
Spend for 40mg/L, sucrose 25g/L, crystal agar 2g/L.
Beneficial effect：Compared with prior art, the preparation method of Salix suchowensis resistance seedling of the invention, it is determined that Salix suchowensis is adopted
With agriculture bacillus mediated transgenosis basic skills and suitable conversion, condition of culture, be forest tree gene functional research and
Genetic improvement provides a new way.
Brief description of the drawings
Fig. 1 is stem section differentiation culture figure；
Fig. 2 is bud elongation culture figure；
Fig. 3 is culture of rootage figure；
Fig. 4 is complete resistant plant figure.
Embodiment
With reference to specific embodiment, the present invention is described further, which can make those skilled in the art
The present invention, but do not limit the invention in any way is more fully understood.
Embodiment 1
Salix suchowensis tissue-cultured seedling successive propagation in a manner of stem section breaks up adventitious bud.Culture minimal medium used is 1/
2WP, sucrose 25-30g/L, pH5.8, crystal agar 2g/L, 121 ± 1 DEG C, sterilize 18min.Additional auxin, cell division
Element and antibiotic are filtration sterilization, add autoclaving postcooling into 40 DEG C or so of culture medium.In subculture about 1 month
The basically identical tender stem segments of growth conditions are chosen in test tube seedling, are cut into the stem section of about 1-2cm long as explant, are inoculated into point
Change preculture in culture medium, as shown in Figure 1.Cultivation temperature is 25 ± 2 DEG C, illumination 3000-4000lux, light application time 16h/d,
Preculture 2-3d.The single bacterium colony of picking Agrobacterium DH5 α is inoculated into LB fluid nutrient mediums, kanamycins 40mg/L is added, in 28
DEG C shaken cultivation (220-230rpm), overnight；Next day takes the LB fluid nutrient mediums of 1mL switchings 50mL.Culture to a certain concentration (into
Enter exponential phase of growth)；5000rpm centrifugations 5-10min collects thalline, takes appropriate WP fluid nutrient mediums suspension thalline to OD600It is worth and is
0.6 or so is spare.Explant stem section after preculture is immersed and infects bacterium solution, ultrasonic wave 10s, slight oscillatory, fully soaks 10-
20min or so, sucks unnecessary bacterium solution on aseptic filter paper, is inoculated on differential medium (being free of antibiotic) and is co-cultured.
After co-culturing 2-4d in the dark, with sterile water washing 6-8 times of cephalosporin is added to remove Agrobacterium, aseptic filter paper sucks unnecessary
Moisture, be inoculated on differential medium (cephalosporin and kanamycins containing respective amount) and cultivate.10d or so, which is replaced, once to be divided
Change culture medium, until differentiating the visible anti-bud of kanamycins, as shown in Figure 2.By kalamycin resistance bud together with former explant one
A bud elongation medium is replaced with Elongation of adventitious bud screening and culturing medium, 20d or so is moved into.It is left to 1-2cm to treat that resistant buds are extended
Each resistant buds are separated independent access screening and culturing medium of taking root and continue to cultivate by the right side, as shown in figure 3, through after a while after
It is commissioned to train to support and can obtain complete resistance seedling, as shown in Figure 4.
Each culture medium used, specific formula are：Minimal medium：1/2WP+ sucrose 25g/L+ crystal agars 2g/L；Winnow with a dustpan
Dustpan willow stem section differential medium：WP+6-BA 0.5mg/L+TDZ 0.005mg/L+NAA0.01mg/L+ sucrose 30g/L+ crystals ocean
Dish 2g/L；Break up screening and culturing medium：Differential medium+cephalosporin (cef) 100mg/L+ kanamycins (kan) 40mg g/L；
The culture medium prescription for being adapted to bud elongation is WP+6-BA 0.5mg/L+IBA 0.01mg/L+ sucrose 30g/L+ crystal agars 2g/L；
Bud extends screening and culturing medium：Bud elongation medium+cephalosporin (cef) 100mg/L+ kanamycins (kan) 40mg/L；It is adapted to life
The culture medium prescription of root is 1/2WP+ sucrose 25g/L+ crystal agars 2g/L；Take root screening and culturing medium：Root media+cephalo is mould
Element (cef) 100mg/L+ kanamycins (kan) 30mg/L.
Embodiment 2
Minimal medium can only maintain the existence of plant and minimum physiological activity demand, it is therefore desirable to cultivate substantially
The basic element of cell division KT and 6-BA are added in base.The preparation method of Salix suchowensis resistance seedling is with embodiment 1, wherein using various concentrations
The basic element of cell division carries out adventitious bud inducing, and each concentration and result are as shown in Table 1 and Table 2.
Influences of 1 6-BA of table to Salix suchowensis adventitious bud inducing
| 6-BA concentration (mg/L) | Bud ratio % | Melting brown rate % |
| 0.1 | 58.9±2.1 | 13.3±0.5 |
| 0.5 | 92.2±3.0 | 14.4±1.3 |
| 1.0 | 74.4±1.7 | 24.4±2.2 |
| 1.5 | 46.7±1.3 | 67.8±1.9 |
| 2.0 | 27.8±0.8 | 84.4±2.7 |
Influences of 2 KT of table to Salix suchowensis adventitious bud inducing
| KT concentration (mg/L) | Bud ratio % | Melting brown rate % |
| 0.1 | 63.3±1.6 | 17.7±2.3 |
| 0.5 | 90.0±1.2 | 15.6±0.4 |
| 1.0 | 65.5±0.7 | 34.4±1.5 |
| 1.5 | 50.0±2.0 | 70.0±1.9 |
| 2.0 | 25.6±1.5 | 87.8±3.7 |
Explant total number × 100% of explant number/inoculation of bud ratio (%)=rudiment.
Explant total number × 100% of explant number/inoculation of melting brown rate (%)=browning.
By that can be drawn to table 1,2 interpretation of result of table, the induction of the basic elements of cell division of various concentrations to adventitious bud has not
Same influence.With the increase of 6-BA concentration, the bud ratio of Salix suchowensis explant is continuously increased, 6-BA concentration reach certain value it
Afterwards, bud ratio starts to reduce, parabolically the rule of formula, while as the increase of 6-BA concentration, the melting brown rate of explant are continuous
Increase；When KT concentration increases, also parabolically formula changes the bud ratio of Salix suchowensis explant, and melting brown rate can increase；Than
Compared with two groups of experimental results, addition 6-BA bud ratios are slightly higher, thus the application addition 6-BA concentration it is optimal be 0.5mg/L.
Embodiment 3
Minimal medium can only maintain the existence of plant and minimum physiological activity demand, it is therefore desirable to cultivate substantially
TDZ and NAA is added in base.The preparation method of Salix suchowensis resistance seedling with embodiment 1, wherein using various concentrations TDZ and NAA into
Row culture, as a result such as table 3.
TDZ, NAA of 3 various concentrations of table combine the influence to adventitious bud proliferation
| TDZ(mg/L) | NAA(mg/L) | Bud growth coefficient |
| 0.001 | 0.01 | 0.90 |
| 0.001 | 0.03 | 1.23 |
| 0.001 | 0.05 | 1.74 |
| 0.003 | 0.01 | 2.31 |
| 0.003 | 0.03 | 2.74 |
| 0.003 | 0.05 | 2.93 |
| 0.005 | 0.01 | 3.48 |
| 0.005 | 0.03 | 3.31 |
| 0.005 | 0.05 | 3.05 |
Bud growth coefficient=propagation explant number/explant sum × 100%.
As known from Table 3, TDZ concentration has adventitious bud proliferation the influence of highly significant, effectively explant can be induced to produce
Raw adventitious bud.NAA promotes cell to uphold, and the growth of bud, the growth to adventitious bud plays an important role.Utilize different TDZ and NAA
Concentration combination can remarkably promote adventitious buds differentiation.Different TDZ and NAA concentration combinations have different promotions to make bud propagation
With the combined concentration that suitable TDZ and NAA are drawn by result is respectively 0.005mg/L, 0.01mg/L.
Embodiment 4
The preparation method of Salix suchowensis resistance seedling is with embodiment 1, wherein being cultivated using different pre-incubation times, as a result such as
Table 4.
Influence of 4 pre-incubation time of table to conversion ratio
| Pre-incubation time (d) | Average conversion (%) |
| 0 | 3.5±0.3 |
| 2 | 8.6±0.6 |
| 4 | 2.2±0.5 |
In test, when being infected without the direct clip explant of preculture, the time of capturing material is restricted, clip
Premature material, which can wilt, influences differentiation, and draws materials again after bacterium solution centrifugation and still need to the long period, so influences OD600It is accurate
Property, feasibility reduces.Meanwhile pollution easily occurs for direct clip material so as to influence to test, and pass through the preculture of 2d, can carry
Early identify the material of pollution.Therefore in actual genetic transformation test, 2d is selected as optimal pre-incubation time.
Embodiment 5
The preparation method of Salix suchowensis resistance seedling is with embodiment 1, wherein being cultivated using different bacterial concentrations, as a result such as table
5。
Influence of 5 bacterial concentration of table to genetic transformation
| Bacterial concentration (OD600) | Average conversion (%) |
| 0.4 | 3.7±0.3 |
| 0.6 | 5.8±0.7 |
| 0.8 | 4.9±0.4 |
The infection ability of the Agrobacterium of different growing stage is different greatly, and the Agrobacterium vigor of increased logarithmic phase is most strong.Bacterium
Liquid excessive concentration can be because subalimentation causes internal competition to aggravate, and the actual Agrobacterium quantity with infection ability is reduced, drop
Low-conversion, while excessive concentration can cause the browning of plant explantation tissue, Agrobacterium is seriously polluted, in the culture in later stage
It is unmanageable.And too low bacterial concentration, thalline quantity are few, the actual thalline with infection ability is less, therefore conversion ratio is low.
As shown in Table 5, the higher bacterial concentration of transformation efficiency is OD600It is worth for 0.6 or so.
Embodiment 6
The preparation method of Salix suchowensis resistance seedling is with embodiment 1, wherein being cultivated using different times of infection, as a result such as table
6。
Influence of 6 time of infection of table to genetic transformation
| Time of infection (min) | Average conversion (%) |
| 10 | 4.5±0.2 |
| 15 | 4.7±0.5 |
| 20 | 4.9±0.4 |
The length of time of infection has the effect of genetic transformation important influence.Research shows that time of infection is long to lure
Plant cell autoimmunity Resistant reaction is led, causes conversion ratio to reduce.But if time of infection is too short, Agrobacterium cannot be effective
Ground infects explant, can equally reduce transformation efficiency, and by interpretation of result, it is 20min to determine optimum time of infection.
Embodiment 7
The preparation method of Salix suchowensis resistance seedling is with embodiment 1, wherein being cultivated using the different co-cultivation times, as a result such as
Table 7.
Table 7 co-cultures influence of the time to genetic transformation
| Co-culture the time (d) | Average conversion (%) |
| 2 | 3.4±0.4 |
| 3 | 6.4±0.4 |
| 4 | 2.6±0.3 |
The co-cultivation time also also has a major impact genetic transformation.Agrobacterium infects entrained external source after plant explant
Gene will not convert immediately, its T-DNA, which is shifted and is incorporated into plant, needs the regular hour.If co-culture time mistake
Short, the increment deficiency of Agrobacterium can cause transformation efficiency to reduce；And co-culture overlong time, then Agrobacterium can be caused excessively raw
It is long, cause plant cell to be drowned, excessive Agrobacterium can block normal absorption of the plant cell to nutritional ingredient, and it is thin to suppress plant
The growth of born of the same parents ultimately results in Plant death.It is preferable to co-culture the time as 3d or so.
Embodiment 8
The preparation method of Salix suchowensis resistance seedling is with embodiment 1, wherein being cultivated using different kanamycins concentration, specifically
It is as follows：
1) concentration of kanamycins during differentiation is cultivated
Differentiation of the kanamycins of various concentrations to explant can produce different influences.In transfer-gen plant resistant buds
In screening, too low kanamycins concentration can cause false positive, increase the workload of later stage Molecular Detection., will in this experiment
The stem section for needing to break up is placed in the differential medium for adding different kan concentration and is cultivated, and is observed after 30d.Analysis knot
Fruit (table 8) understands that, when kanamycins (kan) concentration is 0mg/L, stem section normal differentiation, produces a large amount of adventitious buds and adventitious bud
Upgrowth situation is good；When kanamycins (kan) concentration is 10mg/L, most of stem section can differentiate adventitious bud, differentiation rate
For 46%；When kanamycins (kan) for 20mg/L when, stem section starts yellow occur, and differentiation rate is reduced to 22%；When card, that is mould
When the concentration of plain (kan) increases to 30mg/L, explant stem section yellow is serious, and yellowish-brown is presented, and adventitious bud is considerably less；Further
When increasing the concentration of kanamycins (kan) to 40mg/L, the browning of explant whole is dead.Therefore, the kanamycins of 40mg/L
(kan) concentration can suppress the growth of non-transgenic bud, so selection pressure concentration is kanamycins (kan) 40mg/L.
Influence of the different kanamycins concentration of table 8 to differentiation rate
| Kanamycins concentration (mg/L) | For examination explant number (a) | Break up number (a) | Differentiation rate (%) |
| 0 | 50 | 50 | 100 |
| 10 | 50 | 23 | 46 |
| 20 | 50 | 11 | 22 |
| 30 | 50 | 3 | 6 |
| 40 | 50 | 0 | 0 |
| 50 | 50 | 0 | 0 |
| 60 | 50 | 0 | 0 |
| 70 | 50 | 0 | 0 |
2) take root the concentration of kanamycins
Different vegetable materials are different to the susceptibility of kanamycins, the susceptibility of the different tissues of plant to kanamycins
It is different, it is stronger to the susceptibility of kanamycins for root is compared with other positions, in certain density kanamycins screening and culturing medium
Upper plantation WT lines, it is difficult to take root, and transfer-gen plant then can normally take root.As known from Table 9, kanamycins concentration is
During 0mg/L, adventitious bud can normally take root；When the concentration of kanamycins (kan) is 10mg/L, resistant buds are slow-growing, raw
The adventitious bud quantity of root is largely reduced, rooting rate 42%；When the concentration of kanamycins increases to 20mg/L, adventitious bud is yellow
Change, the adventitious bud quantity taken root is seldom；When the concentration of kanamycins increases to 30mg/L, adventitious bud yellow is withered, and is not taken root.
The kanamycins concentration of 30mg/L can suppress the normal of non-transformed bud and take root, and the adventitious bud converted then can with normal growth,
Therefore, root media see mycin concentration position 30mg/L.
Influence of the different kanamycins concentration of table 9 to taking root
| Kanamycins concentration (mg/L) | For examination explant number (a) | Break up number (a) | Rooting rate (%) |
| 0 | 50 | 50 | 100 |
| 10 | 50 | 21 | 42 |
| 20 | 50 | 4 | 8 |
| 30 | 50 | 0 | 0 |
| 40 | 50 | 0 | 0 |
Embodiment 9
The preparation method of Salix suchowensis resistance seedling is with embodiment 1, wherein being carried out using different cephalosporin class antibiotic concentrations
Culture.Cephalosporin class antibiotic has resistance of wide spectrum, and research shows, its growth to plant cell is poisoned without obvious.This reality
Example selection cephalosporin (cef) is applied as the antibacterial antibiotic in genetic transformation test.Certain density cephalosporin (cef) is right
The Agrobacterium being present in explant surface and shallow layer tissue plays inhibitory action.But the cephalosporin of excessive concentration may shadow
Ring the normal growth of plant.To determine cephalosporin (cef) background concentration, there is provided 4 gradients：0、100、200、300mg/L.
The Agrobacterium EHA105 bacterium solutions containing target gene that 100 μ l have been activated are drawn, are added to the cephalosporin containing various concentrations
(cef) in 3mL LB fluid nutrient mediums, 28 DEG C of constant-temperature table, 220rpm cultures.Cephalosporin (cef) is observed to give birth to Agrobacterium
Long suppression situation, to determine its most effective cephalosporin background concentration.Contain being put into without the Salix suchowensis stem section infected
Cultivated in the screening and culturing medium of different cephalosporin (cef) concentration.4 concentration levels are set：0th, 100,200 and 300mg/L, often
Primary screening culture medium is replaced every 10d, is observed after cultivating 20d.
It is found by experiment that when cephalosporin concentration is 200mg/L, has good inhibition on Agrobacterium and do not influence
The normal growth of explant.
Claims (6)
1. a kind of preparation method of Salix suchowensis resistance seedling, it is characterised in that comprise the following steps：
1）Salix suchowensis tissue-cultured seedling is placed in successive propagation on minimal medium；
2）The tissue-cultured seedling stem of subculture is chosen, is cut into the stem section of 1-2 cm long, cuts off the blade of surrounding, is inoculated in point as explant
Change preculture 2-3d on culture medium；Differential medium is WP culture mediums, additional cellular mitogen 6-BA 0.1-1.0 mg/L, carefully
Born of the same parents' mitogen TDZ 0.005 mg/L, auxin IBA 0.005-0.01mg/L, 30 g/L of sucrose, 2 g/L of crystal agar；
3）The colony inoculation of picking Agrobacterium is into the LB fluid nutrient mediums containing kanamycins, after shaken cultivation, takes part to turn
It is connected in LB fluid nutrient mediums, treats that thalline enters exponential phase of growth, thalline is obtained by centrifugation, takes WP liquid to support base culture medium and hangs
Bacterium solution is infected in floating thalline, acquisition；
4）By step 2）Preculture after stem section immerse step 3）Infect bacterium solution, vibration, after soaking 10-20min, removes more
Remaining bacterium solution, puts back on differential medium and is co-cultured；
5）Agrobacterium is removed to the stem section washing after co-cultivation, is transferred on differentiation screening and culturing medium and cultivates, periodic replacement differentiation
Screening and culturing medium, until differentiating visible kalamycin resistance bud；
6）Kalamycin resistance bud is even transferred on Elongation of adventitious bud screening and culturing medium and continues to cultivate；
7）Treat that resistant buds are extended to 1-2cm, each resistant buds are separated independently, moves into screening and culturing medium of taking root and cultivates, are passed through
The squamous subculture of a period of time can obtain complete resistance seedling；
Step 5）In, the differentiation screening and culturing medium is in differential medium, and it is 200 mg/L to add cephalosporin concentration, card
That mycin concentration is 40 mg/L；
Step 6）In, the Elongation of adventitious bud screening and culturing medium is WP, 0.5 mg/L of additional cellular mitogen 6-BA, auxin
0.01 mg/L of IBA, 200 mg/L of cephalosporin, 40 mg/L of kanamycins；
Step 7）In, the screening and culturing medium of taking root is 1/2WP, and cephalosporin concentration is 100 mg/L, the concentration of kanamycins
For 40 mg/L, 25 g/L of sucrose, 2 g/L of crystal agar.
2. the preparation method of Salix suchowensis resistance seedling according to claim 1, it is characterised in that step 1）In, the base
Basal culture medium is 1/2WP, additional saccharose 25-30 g/L, 5.8,121 ± 1 DEG C of crystal agar 2 g/L, pH, and sterilize 18 min.
3. the preparation method of Salix suchowensis resistance seedling according to claim 1, it is characterised in that step 2）In, described is pre-
Condition of culture is 25 ± 2 DEG C of cultivation temperature, illumination 3000-4000 lux, 16 h/d of light application time.
4. the preparation method of Salix suchowensis resistance seedling according to claim 1, it is characterised in that step 2）In, the differentiation
Culture medium is WP culture mediums, additional cellular mitogen 6-BA 0.5 mg/L, 0.005 mg/L of basic element of cell division TDZ, auxin
0.01 mg/L of IBA, 30 g/L of sucrose, 2 g/L of crystal agar.
5. the preparation method of Salix suchowensis resistance seedling according to claim 1, it is characterised in that step 3）In, the LB
The concentration of kanamycins is 40 mg/L in fluid nutrient medium.
6. the preparation method of Salix suchowensis resistance seedling according to claim 1, it is characterised in that step 3）In, the WP liquid
The OD of body culture medium suspension thalline600It is worth for 0.6.
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| Title |
|---|
| Transcriptome analysis of the differentially expressed genes in the male and femal shrub willows(salix suchowensis);Jingjing Liu et al.;《plos one》;20130401;第8卷(第4期);第1-7页 * |
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Application publication date: 20151021 Assignee: Nanjing Bofei Technology Co., Ltd. Assignor: Nanjing Forestry University Contract record no.: 2019320000120 Denomination of invention: Preparation method of salix suchowensis resistant seedlings Granted publication date: 20180501 License type: Common License Record date: 20190425 Application publication date: 20151021 Assignee: Nanjing Yuanqitong Biotechnology Co., Ltd. Assignor: Nanjing Forestry University Contract record no.: 2019320000121 Denomination of invention: Preparation method of salix suchowensis resistant seedlings Granted publication date: 20180501 License type: Common License Record date: 20190425 Application publication date: 20151021 Assignee: Nanjing biological pest control technology, Limited by Share Ltd Assignor: Nanjing Forestry University Contract record no.: 2019320000122 Denomination of invention: Preparation method of salix suchowensis resistant seedlings Granted publication date: 20180501 License type: Common License Record date: 20190425 |
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