CN112544442A - Method for obtaining fusarium oxysporum-resistant carnation clone - Google Patents
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Abstract
The invention relates to a method for obtaining fusarium oxysporum resistant carnation clone, belonging to the technical field of plant tissue culture. The method utilizes the technology of adding the pathogenic bacteria toxin into the culture medium to screen the disease-resistant germplasm after the cell clone mutagenesis, and can culture and process a large amount of cells in a small space; directly inducing and screening mutants at the cellular level is an attempt for the resistance breeding and the microbiology of higher plants; the method can shorten the disease-resistant breeding period, carries out directional breeding, has high selection efficiency and good effect, saves the field breeding period, and provides a new method for the disease-resistant breeding of the carnation.
Description
Technical Field
The invention belongs to the technical field of plant tissue culture, and particularly relates to a method for obtaining fusarium oxysporum-resistant carnation clone.
Background
Carnation (cartation), also known as Carnation, is a perennial herb of the genus Dianthus (Dianthus Linn) of the family Caryophyllaceae (Caryophyllaceae), is juxtaposed with roses and chrysanthemums as three large cut flowers (guoshao and zhangwei, 1996), accounting for 15% of the sales of fresh cut flowers all over the world. Since the end of the 80's of the 20 th century, carnation had been mass-produced in Yunnan, it occupied a specific gravity of approximately 1/3 in the fresh cut flowers in Yunnan, and became one of the main export fresh cut flowers in Yunnan. According to statistics, the production area of the Yunnan cut-flower carnation in 2014 is 2.84 ten thousand mu, more than 20 hundred million cut-flowers are produced in year, and the main production area is distributed in Kunming, Yuxi, red river, Chuxiong, Qujing and other places.
The problem of plant diseases and insect pests is always the primary factor for restricting the improvement of the production quality of carnation seedlings and cut flowers. Blight caused by fusarium oxysporum f.sp.diathi, a specialized soil-borne fungus of carnation, is one of the most serious soil-borne diseases in the production of carnation (Say, 1992), and infection and damage are reported in the growing region of carnation all over the world. The disease is commonly generated in various carnation production areas in China, has serious harm and difficult prevention and treatment, and causes serious economic loss. Fusarium oxysporum f.sp.mossambucinum mainly damages the roots of carnation plants to cause vascular bundle withering, so that the plants are withered and dead, and the lower branches and leaves of the plants are chlorosis and wilting at the early stage of the disease. Only one side of the plant is affected, and the 'torticollis' symptom is often appeared in the later stage of the disease.
In order to effectively control the blight of the dianthus caryophyllus, researchers at home and abroad study aspects of etiology and biology, prevention and control methods, physiological mechanisms of resistance and the like of the disease for many years, the growth habit, the generation rule and partial physiological mechanisms of the blight of the dianthus caryophyllus are clarified, and breeding and reasonable utilization of disease-resistant varieties are still one of the most effective means in prevention and control. The adoption of the somatic clone variation in vitro screening technology can accelerate the disease-resistant breeding process, but no relevant report is available on carnation at present.
Disclosure of Invention
The invention provides a method for obtaining a fusarium oxysporum-resistant carnation clone, which is convenient to operate and short in period, can obtain the fusarium oxysporum-resistant carnation clone in vitro with an intermediate resistance level, and can be used for disease-resistant breeding of carnations.
In order to realize the purpose, the invention is realized by the following technical scheme:
the method for obtaining the fusarium oxysporum f.sp.bergamot clone specifically comprises the following steps:
1) inducing carnation callus:
inducing stem segments, leaves or stem tips of carnation tissue culture seedlings to obtain calluses; callus can be obtained after general culture for 30d, and then callus with better yellow and loose embryogenesis can be obtained after 2-3 times of subculture;
2) callus suspension culture and EMS mutagenesis:
crushing yellow callus with good loose embryogenesis, and inoculating to a culture medium containing Dicamaba with a concentration of 1.0 mg.L-1Performing shake culture on the suspension culture solution in a conical flask at a constant temperature by a shaking table, and performing suspension culture for 28 days to obtain carnation suspension cell clusters;
preparing EMS solution with the volume percentage concentration of 0.8 percent; selecting carnation suspension cell clusters which basically grow uniformly and are in a yellow loose state, soaking the carnation suspension cell clusters in EMS solution for 4-6 hours, and washing the carnation suspension cell clusters with sterile water for 3 times;
3) pressure screening of fusarium oxysporum toxin:
preparing a toxin selection culture medium containing a fusarium oxysporum toxin extracting solution with a volume fraction of 80%; (ii) a Inoculating the suspending cell mass induced by EMS on a toxin selection culture medium, transferring the surviving cell mass on the toxin selection culture medium without adding toxin after 10d for culture for 10d, transferring the cell mass into a toxin liquid selection culture medium for screening, and repeatedly screening for 3 times in this way;
4) regeneration culture:
transferring the surviving cell mass into a regeneration culture medium for induced differentiation for 30d to obtain the fusarium oxysporum resistant carnation clone in vitro.
Further preferably, in the step 1), the formula of the culture medium for inducing the callus is MS + Dicamaba 0.5-2 mg.L-1+ sugar 30g/L + hydrolyzed casein 300 mg. L-1, pH 5.8.
Further preferably, in the step 1), the formula of the culture medium for inducing the callus is MS + Dicamaba 1 mg.L-1+ sugar 30g/L + hydrolyzed casein 300 mg.L-1And the pH value is 5.8.
Further preferably, in step 3), the toxin selection medium is MS + BA 0.8 mg.L-1+TDZ 0.1mg·L-1+NAA 0.1mg·L-1。
More preferably, in step 4), the regeneration medium is MS + BA 0.5 mg.L-1 + TDZ 0.1 mg.L-1 + NAA 0.1 mg.L-1.
Further preferably, in step 2), the shaking culture conditions of the constant temperature shaking table are 25 ℃ and 120 rpm.
Further preferably, in the step 2), the carnation suspension cell mass 28d is subcultured once, and in the subculture, 1/3 of the old suspension culture solution is poured out and then an equal amount of the new suspension culture solution is added.
The invention has the beneficial effects that:
the method utilizes the technology of adding the pathogenic bacteria toxin into the culture medium to screen the disease-resistant germplasm after the cell clone mutagenesis, and can culture and process a large amount of cells in a small space; directly inducing and screening mutants at the cellular level is an attempt for the resistance breeding and the microbiology of higher plants; the method can shorten the disease-resistant breeding period, carries out directional breeding, has high selection efficiency and good effect, saves the field breeding period, and provides a new method for the disease-resistant breeding of the carnation.
Drawings
FIG. 1 shows callus induction at different parts of a carnation tissue culture seedling, in which callus is formed after 15 days of leaf culture A; b, callus formed after stem tip culture for 20 d; c stem section cultured for 20d to form callus.
FIG. 2 shows the growth of suspension cultures at different sites.
FIG. 3 shows the regeneration of carnation cell mass after pressure screening; a, buds which are just differentiated; b, forming a regeneration plant; c, forming a large number of regeneration plants.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, preferred embodiments of the present invention will be described in detail below to facilitate understanding of the skilled person.
Examples
The test material is a tissue culture seedling of a multi-head variety of the susceptible dianthus caryophyllus, namely purple butterfly. The culture method comprises the following steps:
1) induction of carnation callus
Inducing stem segments, leaves or stem tips of carnation tissue culture seedlings to obtain callus, wherein the formula of a culture medium for inducing the callus is MS + Dicamaba 1 mg.L-1+ sugar 30g/L + hydrolyzed casein 300 mg.L-1The pH value is 5.8; culturing for 30 days to obtain callus, and subculturing for 2-3 times to obtain yellow callus with good loose embryogenesis.
The callus rate of the stem segment, the leaf blade and the stem tip is more than 85%, the callus rate of the leaf blade is the highest and is 94.44%, and the induced callus has good growth and is loose yellow callus (figure 1).
2) Callus suspension culture and EMS mutagenesis
Collecting 2g fresh yellow callus with good loose embryogenesis, crushing with sterilized glass rod, and inoculating to obtain callus containing Dicamaba with concentration of 1.0 mg.L-1Performing shake culture at 25 deg.C and 120rpm on the suspension culture solution in a conical flask, and performing suspension culture for 28d to obtain carnation suspension cell mass; the growth rate was determined by weighing every 7 days. The whole culture process is subcultured once every 28 days, and during subculture, 1/3 old liquid culture medium is poured out and then an equal amount of new culture solution is added.
Preparing EMS solution with the volume percentage concentration of 0.8 percent; selecting carnation suspension cell mass with basically consistent growth (yellow loose state) to be soaked in EMS solution for 4h, and then washing with sterile water for 3 times after treatment; then transferred to a subculture medium (MS + Dicamaba 1 mg. L)-1+ 500 mg.L of hydrolyzed casein-1) Culturing, observing after 20 daysSurvival status of cell pellet and statistical survival.
3) Pressure screening of fusarium oxysporum toxin
Inoculating suspension culture cell mass of multi-head variety purple butterfly of Dianthus caryophyllus mutagenized by EMS into toxin selection culture medium (MS + BA 0.8 mg. L) containing 80% by volume of Fusarium oxysporum toxin extract-1+TDZ 0.1mg·L-1+NAA 0.1mg·L-1) After 10 days, the surviving cell mass was transferred to toxin selection medium (MS + BA 0.8 mg. L)-1+TDZ 0.1mg·L-1+NAA 0.1mg·L-1) Culturing for 10 days, transferring to toxin liquid selection culture medium, and repeatedly screening for 3 times.
After the screening of fusarium oxysporum crude toxin, 13 cell masses of 120 multi-head dianthus caryophyllus varieties purple butterflies survive, the survival rate is 10.83 percent, 3 of the cell masses differentiate adventitious buds, the differentiation rate is 23.08 percent, and the mutation rate is 2.5 percent.
4) And transferring the finally survived cell mass into a regeneration culture medium for inducing differentiation, and counting the differentiation rate after 30 days. The regeneration medium formula is MS + BA 0.5 mg.L-1+TDZ 0.1mg·L-1+NAA 0.1mg·L-1。
Regeneration of the regenerated plants in regeneration medium (BA 0.5 mg. L)-1+TDZ 0.1mg·L-1+NAA 0.1mg·L-1) The medium growth is better, the plant growth is normal, the vitrification phenomenon is avoided, and the plant proliferation is faster.
5) Identification of disease resistance of clone regenerated seedlings of anti fusarium oxysporum
Tissue culture plantlet of multi-head variety purple butterfly of Dianthus caryophyllus and screened disease-resistant regenerated plantlet are artificially inoculated with Dianthus caryophyllus wilt germ spore suspension (spore 1 × 10)6One/ml), 3 replicates, 10 strains per replicate (Fangzhoda, 1998). After 20 days, the selected strain was observed for susceptibility. The disease grading standard is as follows: level 0: no disease symptoms; level 1: the basal leaves were slightly yellow; and 2, stage: the basal leaves are yellow and dry; and 3, level: one side of the branch is bent or withered; 4, level: all shoots present with disease; and 5, stage: and death. The disease resistance degree is divided into 5 types of immunity (I), High Resistance (HR), Medium Resistance (MR), Medium Sensitivity (MS) and High Sensitivity (HS), and the relative disease resistance index is divided into1.00, 0.80-0.99, 0.40-0.79, 0.20-0.39 and less than 0.20. Wherein the relative disease resistance index is 1-relative disease index.
As shown in FIG. 3, the disease resistance of the screened plants was compared with the tissue-cultured plantlets of Oroxylon indicum. After the purple butterfly fusarium oxysporum clone is artificially inoculated with fusarium spore suspension for 10 days, the plant grows normally and shows a certain resistance to fusarium wilt. The relative disease resistance index of the purple butterfly is 0.11, the purple butterfly is a high-susceptible variety, and the relative disease resistance index of the purple butterfly disease resistance clone obtained by toxin pressure screening is 0.45, which is an anti-resistance level.
Analysis of experiments
The test material is a tissue culture seedling of a multi-head variety of the susceptible dianthus caryophyllus, namely purple butterfly.
The Fusarium oxysporum Carcinia specialized (Fusarium oxysporum f.sp. diathi) strain is obtained by separating from Carcinia chinensis wilt strain, and is identified by PCR and conventional culture technology by methods of Wangzhou et al (2004), and is preserved on Potato (PSA) culture medium for later use.
1. Induction of carnation callus
Respectively inducing callus with stem, leaf and stem tip of tissue culture seedling, adding sugar 30g/L, pH 5.8, and hydrolyzing casein 300 mg.L-1The different culture medium formulas are 1, MS and Dicamaba 0.1 mg.L-1，2、MS+Dicamaba 0.3mg·L-1，3、MS+Dicamaba 0.5mg·L-1，4、MS+Dicamaba 1mg·L-1，5、MS+Dicamaba 2mg·L-1. Culturing for 30 days to obtain callus, and subculturing for 2-3 times to obtain yellow callus with good loose embryogenesis.
Callus induction results: better carnation callus is induced, the induction efficiency of different parts of carnation and different concentrations of Dicamaba are compared, the stem, the leaf and the stem tip of the carnation tissue culture seedling are treated by using the Dicamaba with different concentrations, and the results are shown in table 1. The induction rate of stem segments, leaves and stem tips is highest when the concentration of the stem segments, the leaves and the stem tips is 1.0 mg.L < -1 >, the difference with other concentrations of Dicamaba is obvious, the callus rate is more than 85 percent, the callus rate of the leaves is highest and is 94.44 percent, and the induced callus has good growth and is loose yellow callus.
TABLE 1 Effect of Dicamba (Dicamaba) in different concentrations on callus induction in different parts of tissue culture seedlings of purple butterfly
Note: the different lower case letters in the same column were tested for significance at the 0.05 level using the Duncan multiple range test.
2. Callus suspension culture and EMS mutagenesis
2g fresh, loose and friable callus was gently crushed with a sterile glass rod and inoculated into a 100ml Erlenmeba flask with a 40ml Dicamaba concentration of 1.0 mg.L-1The suspension culture solution of (1). The culture was carried out with shaking at 25 ℃ on a 120rpm constant temperature shaker. The growth rate was determined by weighing every 7 days. The whole culture process is subcultured once every 28 days, and during subculture, 1/3 of old suspension culture solution is poured out and then an equal amount of new suspension culture solution is added.
In the initial stage of suspension culture, the suspension culture is granular, subculture is carried out once after 28d, and a suspension culture system is established after 28d through continuous proliferation. When each suspension line was weighed during the suspension culture, the proliferation rate of different suspension lines was found to be different, and the results are shown in FIG. 2. The suspended matter has a growth lag phase in the first 7 days, the proliferation speed of the suspended matter is gradually increased, and the logarithmic phase is reached in 14-28 days, and the nutrition and suspended matter growth environment is proper. After which the growth is more stable, but fresh culture medium needs to be replaced. As can also be seen from FIG. 2, the increase in the rate of the suspension originating from the leaves was most pronounced, and the fresh weight increased to 4.25g after 28 days of suspension culture.
3. EMS mutagenesis
Preparing EMS solution with the volume percentage concentration of 0.2%, 0.4%, 0.6% and 0.8%, taking sterile water without EMS as a control, selecting carnation cell clusters with basically consistent growth, soaking the carnation cell clusters in the EMS solution with each concentration for 1h, 2h, 4h and 6h respectively, washing the carnation cell clusters for 3 times by using sterile water after the treatment, then transferring the carnation cell clusters to a subculture medium for continuous culture, and observing the survival state of the cell clusters after 20 days and counting the survival rate.
The rate of cell pellet survival decreased with increasing treatment time under the same EMS concentration treatment; at the same treatment time, the rate of cell pellet survival decreased as EMS concentration increased. When EMS is used for treatment for 1h, the killing power to cell clusters is weak, physiological damage is not serious, no delay effect is caused, the damage effect of each EMS concentration treatment to embryonic cell clusters is not obvious, and the survival rate is over 60 percent. However, the survival rate tended to decrease with the increase of the treatment time, and when the treatment time was 6 hours, the embryogenic cell masses were browned at each treatment concentration and the survival rate was extremely low. The EMS optimal treatment combination for the cell pellet was finally determined to be 0.4% treatment for 4h based on 50% lethal dose for the pellet treatment.
TABLE 2 survival rate (%)
Note: the different lower case letters in the same column were tested for significance at the 0.05 level using the Duncan multiple range test.
4. Preparation of fusarium oxysporum toxin
The fusarium oxysporum colonies on the potato sucrose culture medium are cut into squares of about 5mm multiplied by 5mm, inoculated into triangular flasks containing 150ml of potato sucrose culture solution, 3 blocks in each flask, placed on a temperature-controlled shaking table, and subjected to shaking culture for 15 days. The shaker speed was 120rpm and the temperature was 25 ℃. When mycelium grows out and the nutrient solution is clear from turbidity, filtering out mycelium and spores by using double-layer gauze, centrifuging the filtrate at 300rpm for 20min, removing precipitate, boiling the supernatant for 15min, cooling, and filtering and sterilizing by using a bacterial filter to obtain a sterile toxin crude extract (Tailianmei, etc., 2005).
5. Determination of Fusarium oxysporum toxin screening pressure
Toxin selection culture medium (MS + BA 0.8 mg.L) containing toxin crude extract with volume fractions of 20%, 40%, 60% and 80% is prepared respectively-1+TDZ0.1mg·L-1+NAA 0.1mg·L-1). Control (CK) toxin selection medium without added toxin. Respectively inoculating the callus of the multi-head variety purple butterfly of carnation to the culture medium, culturing for 10d, observing and counting the survival rate of the callus. Toxin concentrations with a survival rate of 10% -30% were taken as the critical lethal concentrations (charffla, 2005).
As can be seen from Table 3, the toxins all showed inhibitory effects on the growth of carnation calli. With the increase of the volume fraction of the toxin, the survival rate of the callus is reduced, and the inhibition effect is more obvious. The survival rate of the callus is 23.67% under the volume fraction of the 80% toxin crude extract. From the aspect of callus expression state, at 10d, the callus treated by the 80% toxin crude extract is loose, browned and dead, and only a few survives, therefore, the 80% toxin volume fraction treatment of 10d can be regarded as 1 inflection point of most cell growth, in the fusarium oxysporum clone screening test, 80% toxin is critical lethal concentration, and a culture medium containing 80% toxin can be used as a toxin selection culture medium.
TABLE 3 Effect of crude toxin stress on carnation callus survival and desire to grow
Note: the different lower case letters in the same column were tested for significance at the 0.05 level using the Duncan multiple range test.
6. Pressure screening of fusarium oxysporum toxin
Inoculating suspension culture cell mass of multi-head variety purple butterfly of Dianthus caryophyllus after EMS mutagenesis on toxin liquid selective culture medium, transferring the surviving cell mass to liquid suspension subculture medium without toxin after 10d for culture for 10d, transferring to toxin liquid selective culture medium for screening, and repeating the screening for 3 times. And transferring the finally survived cell mass into a regeneration culture medium for inducing differentiation, and counting the differentiation rate after 30 days. The different regeneration culture medium formulas are 1, MS and BA 1.0 mg.L-1+KT 0.1mg·L-1+IBA 0.1mg·L-1，2、MS+BA 1.0mg·L-1+TDZ 0.1mg·L-1+NAA 0.1mg·L-1，3、MS+BA 0.5mg·L-1+KT 0.1mg·L-1+IBA 0.1mg·L-1，4、MS+BA 0.5mg·L-1+TDZ 0.1mg·L-1+NAA 0.1mg·L-1。
After the fusarium oxysporum crude toxin is screened by adopting a one-step selection method, 13 cell masses of 120 multi-head dianthus caryophyllus varieties 'purple butterflies' survive, the survival rate is 10.83 percent, 3 of the cell masses differentiate adventitious buds, the differentiation rate is 23.08 percent, and the mutation rate is 2.5 percent. The above results indicate that the toxin treated cells have been mutated and that a portion of the mutant gene against fusarium toxin is expressed, thereby enabling the cell mass to survive at the critical lethal concentration of fusarium toxin.
In order to improve the regeneration capability and the propagation coefficient of the clone of the anti-fusarium oxysporum, different hormone combinations are adopted to culture the differentiated adventitious buds, and the result shows that the hormone combination with better growth of the regenerated plant is BA 0.5 mg.L-1+TDZ 0.1mg·L-1+NAA 0.1mg·L-1The plant grows normally, the vitrification phenomenon does not exist, and the plant proliferation is fast.
TABLE 4 Effect of different hormone combinations on the regeneration Rate of carnation cell clusters after pressure screening
Note: the different lower case letters in the same column were tested for significance at the 0.05 level using the Duncan multiple range test.
7. Identification of disease resistance of clone regenerated seedlings of anti fusarium oxysporum
Tissue culture plantlet of multi-head variety purple butterfly of Dianthus caryophyllus and screened disease-resistant regenerated plantlet are artificially inoculated with Dianthus caryophyllus wilt germ spore suspension (spore 1 × 10)6One/ml), 3 replicates, 10 strains per replicate (Fangzhoda, 1998). After 20 days, the selected strain was observed for susceptibility. The disease grading standard is as follows: level 0: no disease symptoms; level 1: the basal leaves were slightly yellow; and 2, stage: the basal leaves are yellow and dry; and 3, level: one side of the branch is bent or withered; 4, level: all branch manifestationsSymptoms of the disease; and 5, stage: and death. The disease resistance degree is divided into 5 types of immunity (I), High Resistance (HR), Medium Resistance (MR), Medium Sensitivity (MS) and High Sensitivity (HS), and the relative disease resistance indexes are respectively 1.00, 0.80-0.99, 0.40-0.79, 0.20-0.39 and less than 0.20. Wherein the relative disease resistance index is 1-relative disease index.
The method comprises the steps of inoculating cell masses of a multi-head variety 'purple butterfly' of dianthus caryophyllus subjected to EMS mutagenesis on a culture medium containing a fusarium toxin crude extract with critical lethal concentration, and repeatedly screening to finally obtain the fusarium oxysporum resistant clonal plant. Using mycotoxins as selection agents, care must be taken for appropriate screening concentrations and screening periods, while at the start of the screening, a larger selection population must be available. The adoption of proper toxin concentration is the key of disease-resistant screening, and the high concentration is difficult to obtain a large number of calluses or cells; the concentration is too low, and resistant materials are not easy to screen.
Finally, it is noted that the above-mentioned preferred embodiments illustrate rather than limit the invention, and that, although the invention has been described in detail with reference to the above-mentioned preferred embodiments, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the scope of the invention as defined by the appended claims.
Claims (7)
1. The method for obtaining the fusarium oxysporum f.sp.bergamot clone is characterized by comprising the following steps of: the method specifically comprises the following steps:
1) inducing carnation callus:
inducing stem segments, leaves or stem tips of carnation tissue culture seedlings to obtain calluses; callus can be obtained after general culture for 30d, and then callus with better yellow and loose embryogenesis can be obtained after 2-3 times of subculture;
2) callus suspension culture and EMS mutagenesis:
crushing yellow callus with good loose embryogenesis, and inoculating to a culture medium containing Dicamaba with a concentration of 1.0 mg.L-1Performing shake culture on the suspension culture solution in a conical flask at a constant temperature by a shaking table, and performing suspension culture for 28 days to obtain carnation suspension cell clusters;
preparing EMS solution with the volume percentage concentration of 0.8 percent; selecting carnation suspension cell clusters which basically grow uniformly and are in a yellow loose state, soaking the carnation suspension cell clusters in EMS solution for 4-6 hours, and washing the carnation suspension cell clusters with sterile water for 3 times;
3) pressure screening of fusarium oxysporum toxin:
preparing a toxin selection culture medium containing a fusarium oxysporum toxin extracting solution with a volume fraction of 80%; inoculating the suspending cell mass induced by EMS on a toxin selection culture medium, transferring the surviving cell mass on the toxin selection culture medium without adding toxin after 10d for culture for 10d, transferring the cell mass on the toxin selection culture medium containing 80% toxin for screening, and repeatedly screening for 3 times;
4) regeneration culture:
transferring the surviving cell mass into a regeneration culture medium for induced differentiation for 30d to obtain the fusarium oxysporum resistant carnation clone in vitro.
2. The method of obtaining fusarium oxysporum f.sp.carnation clones as claimed in claim 1, characterized in that: in the step 1), the formula of the culture medium for inducing the callus is MS + Dicamaba 0.5-2 mg.L-1+ sugar 30g/L + hydrolyzed casein 300 mg.L-1And the pH value is 5.8.
3. Method for obtaining anti fusarium oxysporum carnation clones according to claim 1 or 2, characterized in that: in the step 1), the formula of the culture medium for inducing the callus is MS + Dicamaba 1 mg.L-1+ sugar 30g/L + hydrolyzed casein 300 mg.L-1And the pH value is 5.8.
4. Method for obtaining anti-fusarium oxysporum carnation clones according to any of claims 1-3, characterized in that: in the step 3), the toxin selection culture medium is MS + BA 0.8 mg.L-1 + TDZ 0.1 mg·L-1 + NAA 0.1 mg·L-1。
5. Method for obtaining anti-fusarium oxysporum carnation clones according to any of claims 1-4, characterized in that: step 4)In the regeneration medium, MS + BA 0.5 mg.L-1 + TDZ 0.1 mg·L-1 + NAA 0.1 mg·L-1。
6. The method of obtaining fusarium oxysporum f.sp.carnation clones as claimed in claim 1, characterized in that: in the step 2), the shaking culture conditions of the constant temperature shaking table are 25 ℃ and 120 rpm.
7. The method of obtaining fusarium oxysporum f.sp.carnation clones as claimed in claim 1, characterized in that: in the step 2), the carnation suspension cell mass 28d is subcultured once, and in the subculture, 1/3 of old suspension culture solution is poured out and then an equal amount of new suspension culture solution is added.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101810142A (en) * | 2010-04-21 | 2010-08-25 | 云南省农业科学院花卉研究所 | Isolated quick culture method of lily disease resistant plant |
| CN106973791A (en) * | 2017-04-05 | 2017-07-25 | 山西省农业科学院旱地农业研究中心 | A kind of method that ethylmethane sulfonate Vitro Mutation hemerocailis middendorffi produces mutant |
| CN108476981A (en) * | 2018-03-27 | 2018-09-04 | 中国热带农业科学院热带生物技术研究所 | A kind of screening technique of anti-blight banana germplasm |
-
2020
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101810142A (en) * | 2010-04-21 | 2010-08-25 | 云南省农业科学院花卉研究所 | Isolated quick culture method of lily disease resistant plant |
| CN106973791A (en) * | 2017-04-05 | 2017-07-25 | 山西省农业科学院旱地农业研究中心 | A kind of method that ethylmethane sulfonate Vitro Mutation hemerocailis middendorffi produces mutant |
| CN108476981A (en) * | 2018-03-27 | 2018-09-04 | 中国热带农业科学院热带生物技术研究所 | A kind of screening technique of anti-blight banana germplasm |
Non-Patent Citations (6)
| Title |
|---|
| M. THAKUR ET AL.: "In vitro selection and regeneration of carnation (Dianthus caryophyllus L.) plants resistant to culture filtrate of Fusarium oxysporum f.sp. dianthi", 《PLANT CELL REP》 * |
| 彭绿春等: "应用尖孢镰刀菌培养滤液室内鉴定香石竹品种抗病性初探", 《江西农业学报》 * |
| 敖世恩等: "水稻抗纹枯病突变体的离体筛选", 《华南农业大学学报》 * |
| 罗静等: "EMS 离体诱变及抗草莓灰霉病愈伤组织的筛选", 《核农学报》 * |
| 郭丽娟等: "通过组织培养筛选小麦抗赤霉病突变体的研究", 《遗传学报》 * |
| 钱丽娟等: "不同激素配比对香石竹茎尖组织培养的影响", 《农业科学研究》 * |
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