CN115191350B - Method for inducing regeneration of adventitious buds of stem explant of sedum aizoon - Google Patents
Method for inducing regeneration of adventitious buds of stem explant of sedum aizoon Download PDFInfo
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Abstract
The invention discloses a method for inducing adventitious buds of a stem explant of zoysia ferruginea. The invention creatively discovers the promoting effect of copper ions, magnesium ions, silver ions and nitroprusside on the adventitious bud regeneration of the stem explant of the armored grass, develops and optimizes the optimal culture medium and culture conditions for artificial culture of the armored grass, establishes a rapid and effective in-vitro propagation regeneration system of the stem explant of the armored grass, simplifies the tissue culture method, shortens the tissue culture period and improves the quality of regenerated seedlings; provides a method and a basis for realizing the industrial cultivation of the armored exact plant, and has certain practical significance for the increasing market demand of the armored exact plant in Guangdong.
Description
Technical Field
The invention belongs to the technical field of plant cell engineering, and particularly relates to a method for inducing regeneration of adventitious buds of a stem explant of zoysia ferruginea.
Background
Armored (Cassia mimosoides Linn.), also known as Cassia occidentalis, is a plant of the Cassia genus of the Leguminosae family (Leguminosae), and is a perennial shrub-like mantle herb. Techloa sellifolia is now distributed in tropical and subtropical regions of the world, and southeast, south and southwest of China are regions in which it is widely distributed. The armored plant species usually grows in wastelands, is drought-tolerant and barren-tolerant, can be used as green manure, and is a good covering plant and a soil-improved plant. The Tiejia Cao is originally recorded in the Ming Dynasty 'Jiu Dian Ben Cao', can be used as a medicine by using the whole herb, has the effects of tonifying spleen, promoting diuresis, clearing heat, removing toxicity, relaxing bowels and the like, and can be used as tea for young stem leaves and leaves, and the roots can treat dysentery. Due to its special medicinal value, it has been widely spread in folks for hundreds of years. Because the armored grass has important medicinal value, the demand of the armored grass on the market is increasing day by day. However, in natural environment, the gerbera ferruginea has various problems that the germination rate of seeds is low, the planting steps are complicated, the natural propagation period is long, the excellent quality of a mother plant is difficult to store, and the like.
At present, few research reports about the tissue culture regeneration of the sedum aizoon are reported, the influence of different types of hormones or hormones with different concentrations on the tissue culture regeneration effect of the sedum aizoon explant is generally concerned, the reported period of the adventitious bud regeneration culture of the sedum aizoon explant is long, generally at least 80 days, complicated processes such as proliferation culture and elongation culture are needed, and the efficiency of large-scale propagation expansion of the sedum aizoon is severely limited.
Disclosure of Invention
The invention aims to provide a method for efficiently inducing an explant of a stem section of sedum aizoon to generate regenerated adventitious buds, aiming at the problems of low induction rate, poor quality, long period and the like in the processes of sedum aizoon adventitious bud induction and plant regeneration in the prior art.
In order to improve the quality and efficiency of inducing adventitious bud formation of the stem explant of the sedum aizoon, the invention creatively adopts metal ions with different concentrations and different types to induce the stem explant to generate the adventitious bud, and the optimized conditions can obviously improve the efficiency and quality of callus induction. Then inoculating the obtained regenerated bud sticks to rooting culture media added with different types of additives for culturing for a period of time, and obtaining complete regenerated plants.
Therefore, the invention aims to provide a method for inducing regeneration of adventitious buds of a stem explant of sedum aizoon, which comprises the following steps:
a. taking the armored seeds, carrying out germination accelerating treatment and disinfection treatment, and then inoculating the seeds on an MS solid culture medium for aseptic germination culture;
b. taking a gerbera acuminata sterile plant growing for 15-25 days, removing roots and leaves, and keeping stems; cutting the stem into small segments as stem explant;
c. axially and horizontally inoculating the stem section explants to an adventitious bud induction culture medium, and carrying out induction culture on adventitious buds;
d. and (3) inoculating the adventitious buds with the length of more than 1cm to a rooting culture medium, and inducing the adventitious buds to root to obtain the regenerated plantlets of the armored grass.
Preferably, the germination accelerating treatment in the step a is as follows: soaking the seeds in 80 deg.C water for 10min.
Preferably, the sterilization treatment of step a is: sterilizing with 75% ethanol water solution for 30-45s, sterilizing with 2.5% NaClO water solution for 10-15min, shaking, and washing with sterilized water for 1min for 3-4 times.
Preferably, the MS solid medium comprises: 30g/L of sucrose and 8g/L of agar, and the balance of MS culture medium, and the pH value is 5.8-6.0.
Preferably, the cutting of the stem into small segments in step b is cutting the stem into small segments of about 0.5cm in length.
Preferably, the adventitious bud induction medium contains: TDZ 0.5mg/L, KT 2mg/L, nitroprusside 1.2mg/L, agCl 0.3mg/L, sucrose 30g/L and agar 8g/L, the balance being MS culture medium, pH 5.8-6.0.
Preferably, the adventitious bud induction medium contains: TDZ 0.5mg/L, KT 2mg/L, cuCl 2 0.3mg/L、MgSO 4 ·7H 2 0.5mg/L of O, 30g/L of cane sugar and 8g/L of agar, and the balance is MS culture medium with pH of 5.8-6.0.
Preferably, the adventitious bud induction medium contains: TDZ 0.5mg/L, KT 2mg/L, feSO 4 ·7H 2 0.4mg/L of O, 30g/L of cane sugar and 8g/L of agar, and the balance of MS culture medium, and pH value is 5.8-6.0.
Preferably, the rooting medium comprises: IBA 0.1mg/L, DA-6.6 mg/L, sucrose 30g/L, agar 8g/L, and MS culture medium in balance, with pH 5.8-6.0.
Preferably, the method for inducing the adventitious bud regeneration of the stem explant of the sedum aizoon comprises the following culture conditions: the illumination intensity is 2000lx, the illumination time is 12 hours/day, and the temperature is 25 +/-1 ℃.
The method optimized by the invention can obviously improve the efficiency and quality of inducing the formation of the regenerated adventitious buds of the tuber segment explant of the sedum aizoon and lays a good work foundation for the asexual rapid propagation and the fine variety breeding of the tuber segment explant of the sedum aizoon.
The invention has the beneficial effects that:
the invention takes the common liver-protecting Chinese herbal medicine of the armored grass in Guangdong area as an object, creatively discovers the promoting effect of copper ions, magnesium ions, silver ions and sodium nitroprusside on the adventitious bud regeneration of the armored grass stem explant by applying a plant tissue culture technology, develops and optimizes the optimal culture medium and culture conditions for artificial culture of the armored grass, establishes a rapid and effective armored grass stem explant in-vitro propagation regeneration system, simplifies a tissue culture system, shortens a tissue culture period and improves the quality of regenerated seedlings. The method provides a method for breeding and storing the germplasm resources of the armored grass, and provides raw material support for further understanding basic researches such as pharmacological activity analysis and liver protection application of the armored grass; provides a method and a basis for realizing the industrial cultivation of the armored exact plant, and has certain practical significance for the increasing market demand of the armored exact plant in Guangdong.
The method can obviously shorten the time for obtaining the complete regeneration plant of the sedum aizoon, does not need the steps of propagation culture, elongation culture and the like, and simplifies the tissue culture system. Meanwhile, the regenerated adventitious bud obtained by the method has better quality, and the shoot rooting culture can be directly carried out after the adventitious bud induction of the stem explant, so that a complete regenerated plant can be obtained within 60 days, the regeneration efficiency is obviously improved, and the tissue culture period is shortened.
Drawings
FIG. 1 shows the effect of adventitious bud regeneration in a shoot explant of Tephrosia purpurea, wherein (A) the effect of a control obtained by directly inoculating a shoot explant of Tephrosia purpurea onto a hormone-free MS solid medium for 40 days; directly inoculating the stem explant axially and horizontally to an MS solid culture medium added with 2mg/L KT (B) and 0.5mg/L TDZ (C) for adventitious bud induction culture for 40 days; using MS solid culture medium containing 0.5mg/L TDZ and 2mg/L KT as basic culture medium, respectively axially and horizontally inoculating the obtained stem section explants to the culture medium containing 0.3mg/L CuCl 2 (D)、0.5mg/L MgSO 4 ·7H 2 O(E)、0.4mg/L FeSO 4 ·7H 2 Performing adventitious bud induction culture on a basal medium containing O (F), 1.2mg/L of sodium nitroprusside (G) and 0.3mg/L of AgCl (H) for 40 days; the basic culture medium is added with (I) 12mg/L of sodium nitroprusside and 0.3mg/L of CuCl 2 The adventitious bud regeneration effect is good; (J) 0.3mg/L AgCl and 0.4mg/L FeSO are added into a basic culture medium 4 ·7H 2 Adventitious bud regeneration effect at O; using MS solid culture medium containing 0.5mg/L TDZ and 2mg/L KT as basal medium, axially and horizontally inoculating the obtained stem segment explant on the basal medium containing 1.2mg/L nitroprusside for adventitious bud induction culture for 40 days, vertically inoculating regenerated bud stick with length more than 1cm (morphological lower end downwards) on the basal medium added with 0.6mg/L DA-6 (K) and 0.4mg/L H 3 BO 3 Effect of rooting induction culture in (L) rooting basal medium (MS solid medium +0.1mg/L IBA) for 20 days (bar =1 cm).
Detailed Description
The following examples are further illustrative of the present invention and are not intended to be limiting thereof.
Example 1
1. Reagent and method
1. Required main reagent
Kinetin (KT), thidiazuron (TDZ), anhydrous copper chloride (CuCl) 2 ) Magnesium sulfate heptahydrate (MgSO) 4 ·7H 2 O), ferrous sulfate (FeSO) 4 ·7H 2 O), nitroprusside (SNP), silver chloride (AgCl), diethyl aminoethyl hexanoate (DA-6), boric acid (H) 3 BO 3 ) And indolebutyric acid (IBA) are analytically pure reagents, and are provided by Nanjing reagent (Nanjing chemical reagents, inc.). MS bulk powder is supplied by Solebao (Beijing).
Preparation of MS solid Medium
The MS solid culture medium used in this example was MS culture medium + sucrose 30g/L + agar 8g/L. The MS medium is a general medium with a formula known in the art, and the components of the medium are Murashige T, skoog F (1962) A recycled medium for rapid growth and bio assays with a tobaco tissue cultures, physiologia Plantarum 15.
The preparation method in the implementation is as follows:
MS solid culture medium: 4g/L of MS bulk powder, 1mL of MS trace (1000 Xmother liquor), 10mL of MS organic (100 Xmother liquor), 10mL of Fe salt (100 Xmother liquor), 100mg/L of inositol, 30g/L of sucrose and 8g/L of agar.
According to the required culture dosage, respectively weighing a large amount of MS powder, inositol and sucrose by using an electronic balance, respectively extracting MS trace, organic and Fe salts by using a liquid-transferring gun, adding the weighed medicament into a prepared beaker, adding a proper amount of distilled water, and fully dissolving the medicament by using a constant-temperature magnetic stirrer. The volume of the dissolved solution is adjusted to 1L, and the pH of the solution is adjusted to 5.8 by using a pH meter, sodium hydroxide (NaOH) and hydrochloric acid (HCl). Weighing 8g of agar powder by using an electronic balance, pouring the solution into a pot, heating to 50-60 ℃, pouring weighed agar powder, continuously stirring to fully dissolve the agar powder in the pot, heating to boiling, and subpackaging the obtained mixture into prepared tissue culture bottles. Sterilizing the culture medium and the cutting plate in a high pressure steam sterilizing pot (121 deg.C, 0.1 MPa) for 20 min, taking out, and cooling at room temperature for solidification.
3. Germination of Meadowrum caudatum seeds
Soaking a certain amount of Medinioides Sempervirens seeds in 80 deg.C water for 10min, sterilizing with 75% ethanol for 30-45s, sterilizing with 2.5% sodium hypochlorite (NaClO) water solution for 10-15min, shaking continuously, washing with sterilized distilled water in a superclean bench for 3-4 times (1 min each time), and inoculating to MS solid culture medium for aseptic germination culture.
4. Obtaining the explant of the stem segment of the sedum aizoon
Taking a gerbera acuminata sterile plant which grows for 20 days, removing roots and leaves, reserving stems, and cutting the stems into small sections (without buds) of about 0.5cm to serve as stem explants.
5. Inducing adventitious bud regeneration of sedum aizoon stem explant
5.1 Using conventional methods
(1) The obtained stem explant is directly and horizontally inoculated on MS solid culture medium added with KT (0, 0.25, 0.5, 1 and 2 mg/L) with different concentrations in an axial direction for adventitious bud induction culture for 40 days, and the culture results under different conditions are compared.
(2) The obtained stem explant is directly inoculated on MS solid culture medium added with TDZ (0, 0.25, 0.5, 1 and 2 mg/L) with different concentrations axially and horizontally for adventitious bud induction culture for 40 days, and the culture results under different conditions are compared.
5.2 New Process
Using MS solid culture medium containing 0.5mg/L TDZ and 2mg/L KT as basic culture medium, respectively axially and horizontally inoculating the obtained stem section explants to explants containing copper ions (CuCl) with different concentrations 2 0, 0.3, 0.6, 0.9 and 1.2 mg/L), magnesium ion (MgSO 4 ·7H 2 O is 0, 0.25, 0.5, 0.75 and 1.5 mg/L), ferrous ion (FeSO) 4 Adventitious bud induction culture was performed for 40 days on a basal medium of 7H2O at 0, 0.2, 0.4, 0.8, and 1.6 mg/L), nitroprusside (0, 0.3, 0.6, 1.2, and 2.4 mg/L), silver ion (AgCl at 0, 0.15, 0.3, 0.6, and 1.2 mg/L), and the culture results under different conditions were compared.
6. Rooting induction of regenerated shoot
Using MS solid culture medium containing 0.5mg/L TDZ and 2mg/L KT as basal medium, axially and horizontally inoculating the obtained stem segment explants onto the basal medium containing 1.2mg/L nitroprusside for adventitious bud induction culture for 40 days, vertically inoculating regenerated bud strips (adventitious buds) with the length of more than 1cm (with morphological lower ends downwards) onto the basal medium added with DA-6 (0, 0.15, 0.3, 0.6 and 1.2 mg/L) and boron ions (H) with different concentrations 3 BO 3 0, 0.2, 0.4, 0.8 and 1.6 mg/L) in rooting basal medium (MS solid medium +0.1mg/L IBA) for 20 days, and the culture results under different conditions were compared.
7. Culture conditions and data analysis
All experiments were performed under the same conditions. The pH of the medium was adjusted to 5.8 using 1mol/L HCl solution or 1mg/L NaOH solution, and then autoclaved at 121 ℃ under 0.1MPa for 15min. The culture conditions in the tissue culture chamber are 2000lx illumination intensity, 12 h/day illumination time and 25 +/-1 ℃ culture temperature. All experimental treatments were repeated three times, the experimental data are represented by the mean ± standard deviation of 3 repeated experiments, all data were analyzed by SPSS Statistics 17.0 statistical analysis software for anova and duncan multiple comparisons (P ≦ 0.05), and the difference in letters after data indicates significant differences between treatments.
2. Results and description
The regeneration effect of adventitious buds of the explants of the stem segments of the Meadowrum distichum is partially shown in figure 1.
1. Influence of KT with different concentrations on regeneration effect of stem explant of Mesona chinensis Benth
In the experiment, a conventional method is adopted, the obtained stem section explants are directly and axially horizontally inoculated to MS solid culture media only added with KT (0, 0.25, 0.5, 1 and 2 mg/L) with different concentrations for adventitious bud induction culture for 40 days, and the results in Table 1 show that the adventitious bud obtained by adopting the method has low regeneration efficiency and poor quality of regenerated adventitious buds. When the concentration of KT added to the medium is 2mg/L, the best adventitious bud induction effect can be obtained, the regeneration rate of the adventitious buds reaches the maximum value of 25.93%, the average bud number also reaches the maximum value of 2.37, and the average bud length also reaches the maximum value of 0.76cm. The effect of adventitious bud regeneration will become worse with the further increase of the concentration of KT.
TABLE 1 Effect of KT at different concentrations on the regeneration Effect of shoot explants of Trifolium mexicana
Note: data were analyzed for variance and Duncan multiple comparisons (P ≦ 0.05) using SPSS Statistics 17.0 statistical analysis software, with the difference in letters after the data indicating significant differences between treatments.
2. Influence of TDZ with different concentrations on regeneration effect of stem explant of Mesona chinensis Benth
In the experiment, a conventional method is adopted, the obtained stem explant is directly and axially horizontally inoculated to MS solid culture media added with TDZ (0, 0.25, 0.5, 1 and 2 mg/L) with different concentrations for adventitious bud induction culture for 40 days, and the results in a table 2 show that the adventitious bud obtained by adopting the method is low in regeneration efficiency and poor in quality of regenerated adventitious buds. When the TDZ concentration added to the medium was 0.5mg/L, the optimum adventitious bud induction effect was obtained, and at this time, the adventitious bud regeneration rate reached a maximum of 18.91%, the average number of buds reached a maximum of 1.67, and the average bud length reached a maximum of 0.46cm. The effect of adventitious bud regeneration will become worse by continuing to increase the concentration of TDZ.
TABLE 2 influence of TDZ of different concentrations on regeneration effect of armored stem explants
| TDZ concentration (mg/L) | Regeneration ratio (%) | Average number of buds | Average bud Length (cm) |
| 0 | 0±0e | 0±0d | 0±0d |
| 0.25 | 11.47±1.13b | 1.23±0.15b | 0.23±0.04b |
| 0.5 | 18.91±0.57a | 1.67±0.28a | 0.46±0.05a |
| 1 | 8.49±1.15c | 0.85±0.12c | 0.19±0.02bc |
| 2 | 5.77±0.71d | 0.65±0.08c | 0.13±0.03c |
Note: data were analyzed for variance and Duncan multiple comparisons (P ≦ 0.05) using SPSS Statistics 17.0 statistical analysis software, with the difference in letters after the data indicating significant differences between treatments.
3. Cu of different concentrations 2+ Influence on regeneration effect of stem explant of Carex meyeriana
The experiment adopts a new method, takes MS solid culture medium containing 0.5mg/L TDZ and 2mg/L KT as basic culture medium, and inoculates the obtained stem explant axially and horizontally to contain copper ions (added CuCl) with different concentrations 2 0, 0.3, 0.6, 0.9 and 1.2 mg/L) on a minimal medium.
Table 3 the results show that CuCl was added to the basal medium 2 The best adventitious bud induction effect can be obtained when the concentration is 0.3mg/L, the regeneration rate of the adventitious buds reaches a maximum value of 55.56%, the average bud number also reaches a maximum value of 4.97, and the average bud length also reaches a maximum value of 1.88cm. The effect of adventitious bud regeneration will deteriorate as the concentration of copper ions continues to increase.
TABLE 3 Cu concentrations 2+ Influence on regeneration effect of stem explant of Meadowrue Diels
| CuCl 2 Concentration (mg/L) | Regeneration ratio (%) | Average number of buds | Average bud Length (cm) |
| 0 | 28.22±1.35d | 2.55±0.08d | 0.93±0.04d |
| 0.3 | 55.56±1.06a | 4.97±0.23a | 1.88±0.07a |
| 0.6 | 45.84±1.71b | 4.13±0.15b | 1.49±0.06b |
| 0.9 | 34.56±0.93c | 2.98±0.18c | 1.32±0.06c |
| 1.2 | 22.53±1.10e | 2.32±0.14d | 1.05±0.15d |
Note: data were analyzed for ANOVA and Duncan multiple comparisons (P ≦ 0.05) using SPSS statics 17.0 statistical analysis software, with differences in letters after the data indicating significant differences between treatments.
4. Different concentrations of Mg 2+ For the explant of the stem segment of the armored grassInfluence of the biological Effect
The experiment adopts a new method, takes MS solid culture medium containing 0.5mg/L TDZ and 2mg/L KT as basic culture medium, and inoculates the obtained stem explant axially and horizontally to contain magnesium ions (added MgSO) 4 ·7H 2 O0, 0.25, 0.5, 0.75 and 1.5 mg/L) in a basal medium.
Table 4 the results show that MgSO was added when basal medium was added 4 ·7H 2 When the O concentration is 0.5mg/L, the best adventitious bud induction effect can be obtained, the regeneration rate of the adventitious buds reaches a maximum value of 52.68%, the average bud number also reaches a maximum value of 4.18, and the average bud length also reaches a maximum value of 1.51cm. The effect of adventitious bud regeneration will be deteriorated by continuing to increase the concentration of magnesium ions.
TABLE 4Mg at various concentrations 2+ Influence on regeneration effect of stem explant of Carex meyeriana
| MgSO 4 ·7H 2 O concentration (mg/L) | Regeneration rate (%) | Average number of buds | Average bud Length (cm) |
| 0 | 27.91±1.36c | 2.54±0.06c | 0.91±0.08c |
| 0.25 | 45.86±1.72b | 3.39±0.22b | 1.32±0.07b |
| 0.5 | 52.68±1.84a | 4.18±0.17a | 1.51±0.06a |
| 0.75 | 43.00±2.14b | 3.44±0.23b | 1.25±0.07b |
| 1.5 | 29.20±1.43c | 2.55±0.12c | 1.03±0.11c |
Note: data were analyzed for ANOVA and Duncan multiple comparisons (P ≦ 0.05) using SPSS statics 17.0 statistical analysis software, with differences in letters after the data indicating significant differences between treatments.
5. Different concentrations of Fe 2+ Influence on regeneration effect of stem explant of Carex meyeriana
The experiment adopts a new method, takes MS solid culture medium containing 0.5mg/L TDZ and 2mg/L KT as basic culture medium, and inoculates the obtained stem explant axially and horizontally to contain ferrous ions (added FeSO) with different concentrations 4 ·7H 2 O is 0, 0.2, 0.4, 0.8 and 1.6 mg/L) on a basal medium.
Table 5 results show that FeSO was added to the basal medium 4 ·7H 2 When the concentration of O is 0.4mg/L, the best adventitious bud induction effect can be obtained, the regeneration rate of the adventitious buds reaches the maximum value of 41.87 percent, the average bud number also reaches the maximum value of 2.96, and the average bud length also reaches the maximum value of 1.38cm; while the regeneration obtained at this time is indefiniteThe quality of the bud is poor, and the leaves are easy to turn yellow and shrink. The effect of adventitious bud regeneration will become worse by continuing to increase the ferrous ion concentration.
TABLE 5 different concentrations of Fe 2+ Influence on regeneration effect of stem explant of Meadowrue Diels
| FeSO 4 ·7H 2 O concentration (mg/L) | Regeneration ratio (%) | Average number of buds | Average bud Length (cm) |
| 0 | 28.17±1.88d | 2.57±0.07b | 0.96±0.08c |
| 0.2 | 36.17±2.21b | 2.85±0.1a | 1.19±0.10b |
| 0.4 | 41.87±3.65a | 2.96±0.16a | 1.38±0.07a |
| 0.8 | 34.18±2.25bc | 2.84±0.08a | 1.29±0.06ab |
| 1.6 | 30.50±2.16cd | 2.51±0.15b | 0.85±0.05c |
Note: data were analyzed for variance and Duncan multiple comparisons (P ≦ 0.05) using SPSS Statistics 17.0 statistical analysis software, with the difference in letters after the data indicating significant differences between treatments.
6. Influence of SNP (single nucleotide polymorphism) with different concentrations on regeneration effect of calliopsis bodinieri stem explant
In the experiment, a new method is adopted, MS solid culture medium containing 0.5mg/L TDZ and 2mg/L KT is used as a basic culture medium, and the obtained stem explant is axially and horizontally inoculated on the basic culture medium containing different concentrations of SNP (0, 0.3, 0.6, 1.2 and 2.4 mg/L) for adventitious bud regeneration induction.
The results in Table 6 show that the optimum adventitious bud induction effect can be obtained when the concentration of SNP added to the basal medium is 1.2mg/L, and that the adventitious bud regeneration rate reaches a maximum of 66.19%, the average bud number also reaches a maximum of 5.44, and the average bud length also reaches a maximum of 3.46cm. The effect of adventitious bud regeneration will be worse by continuing to increase the concentration of nitroprusside.
TABLE 6 influence of SNP on the regeneration effect of the shoot explants of Carex meyeriana
| SNP concentration (mg/L) | Regeneration ratio (%) | Average number of buds | Average bud Length (cm) |
| 0 | 29.19±1.55d | 2.59±0.04e | 1.01±0.06d |
| 0.3 | 45.28±2.38c | 3.57±0.31d | 1.71±0.15c |
| 0.6 | 52.79±1.60b | 4.76±0.12b | 3.17±0.08b |
| 1.2 | 66.19±3.18a | 5.44±0.18a | 3.46±0.09a |
| 2.4 | 54.53±2.84b | 4.27±0.11c | 2.98±0.14b |
Note: data were analyzed for variance and Duncan multiple comparisons (P ≦ 0.05) using SPSS Statistics 17.0 statistical analysis software, with the difference in letters after the data indicating significant differences between treatments.
7. Influence of silver ions with different concentrations on regeneration effect of armored breaker stem explant
In the experiment, a new method is adopted, MS solid culture medium containing 0.5mg/L TDZ and 2mg/L KT is used as a basic culture medium, and the obtained stem explant is horizontally inoculated on the basic culture medium containing silver ions with different concentrations (the added AgCl is 0, 0.15, 0.3, 0.6 and 1.2 mg/L) to carry out adventitious bud regeneration induction.
The results in Table 7 show that the best adventitious bud induction effect was obtained when the AgCl concentration added to the basal medium was 1.2mg/L, and that the adventitious bud regeneration rate reached a maximum of 63.96%, the average bud number also reached a maximum of 5.39, and the average bud length also reached a maximum of 3.49cm. The effect of adventitious bud regeneration will deteriorate as the concentration of silver ions continues to increase.
TABLE 7 Effect of different concentrations of Ag + on the regeneration of Mesona stem explants
| AgCl concentration (mg/L) | Regeneration rate (%) | Average number of buds | Average bud Length (cm) |
| 0 | 29.95±1.49e | 2.65±0.05d | 0.98±0.08d |
| 0.15 | 48.36±2.42c | 3.53±0.11c | 1.69±0.09c |
| 0.3 | 63.96±1.90a | 5.39±0.31a | 3.49±0.17a |
| 0.6 | 52.95±2.86b | 4.27±0.17b | 3.07±0.12b |
| 1.2 | 37.72±2.96d | 3.38±0.21c | 1.66±0.13c |
Note: data were analyzed for variance and Duncan multiple comparisons (P ≦ 0.05) using SPSS Statistics 17.0 statistical analysis software, with the difference in letters after the data indicating significant differences between treatments.
8. Influence of different culture medium component combinations on adventitious bud regeneration effect of armored grass stem explant
The resulting stem explants were inoculated axially and horizontally to a medium containing 7 different combinations of ingredients (combination 1, 0.3mg/L AgCl and 0.3mg/L CuCl, using MS solid medium containing 0.5mg/L TDZ and 2mg/L KT as basal medium for this experiment 2 (ii) a And (3) combination 2:0.3mg/L AgCl and 0.5mg/L MgSO 4 ·7H 2 O; and (3) combination: 0.3mg/L AgCl and 0.4mg/L FeSO 4 ·7H 2 O; and (4) combination: 1.2mg/L of sodium nitroprusside and 0.3mg/L of CuCl 2 (ii) a And (3) combination 5:1.2mg/L sodium nitroprusside and 0.5mg/L MgSO 4 ·7H 2 O; and (4) combination 6:1.2mg/L of sodium nitroprusside and 0.4mg/L of FeSO 4 ·7H 2 O; combination 7:1.2mg/L nitroprusside and 0.3mg/L AgCl).
The results in Table 8 show that when 1.2mg/L nitroprusside and 0.3mg/L CuCl were added to the basal medium 2 Then, the best adventitious bud induction effect can be obtained, the regeneration rate of the adventitious buds reaches the maximum value of 76.21%, and the average bud numberAlso reached a maximum of 5.64 particles, and the average shoot length also reached a maximum of 3.71cm. When 0.3mg/L AgCl and 0.4mg/L FeSO are added into the basic culture medium 4 ·7H 2 O, the adventitious bud regeneration induction effect was the worst, and the adventitious bud regeneration rate was the minimum (44.81%), the average bud number was the minimum (2.99), and the average bud length was the minimum (1.41 cm) in comparison with the case where FeSO alone was added 4 ·7H 2 The quality of the regenerated adventitious bud obtained in O is slightly better, and the shape of the leaf is normal.
TABLE 8 Effect of different combinations of ingredients on the regeneration of adventitious buds of a shoot explant of Carex meyeriana
Note 1: data were analyzed for variance and Duncan multiple comparisons (P ≦ 0.05) using SPSS Statistics 17.0 statistical analysis software, with the difference in letters after the data indicating significant differences between treatments.
Note 2: on adventitious bud regeneration induction basal medium (MS solid medium added with 0.5mg/L TDZ and 2mg/L KT), combination 1:0.3mg/L AgCl and 0.3mg/L CuCl 2 (ii) a And (3) combination 2:0.3mg/L AgCl and 0.5mg/L MgSO 4 ·7H 2 O; and (3) combination: 0.3mg/L AgCl and 0.4mg/L FeSO 4 ·7H 2 O; and (4) combination: 1.2mg/L of sodium nitroprusside and 0.3mg/L of CuCl 2 (ii) a And (3) combination 5:1.2mg/L sodium nitroprusside and 0.5mg/L MgSO 4 ·7H 2 O; and (4) combination 6:1.2mg/L of sodium nitroprusside and 0.4mg/L of FeSO 4 ·7H 2 O; combination 7:1.2mg/L of sodium nitroprusside and 0.3mg/L of AgCl.
9. Influence of DA-6 with different concentrations on rooting effect of regenerated shoot of explant of stem segment of Mesona chinensis Benth
Taking an MS solid culture medium containing 0.5mg/L TDZ and 2mg/L KT as a basic culture medium, axially and horizontally inoculating the obtained stem segment explants onto the basic culture medium containing 1.2mg/L nitroprusside for adventitious bud induction culture for 40 days, vertically inoculating regenerated bud sticks (adventitious buds) with the length of more than 1cm (the lower end of morphology is downward) onto a rooting medium base (MS solid culture medium +0.1mg/L IBA) added with different concentrations of DA-6 (0, 0.15, 0.3, 0.6 and 1.2 mg/L) for adventitious bud induction culture for 20 days, and finding that the best rooting effect can be obtained by adding 0.6mg/L DA-6 into the rooting basic culture medium.
The results in Table 9 show that the optimum rooting effect can be obtained when the concentration of DA-6 added to the rooting basal medium is 0.6mg/L, the rooting rate reaches a maximum of 90.23%, the average number of roots also reaches a maximum of 6.08, and the average root length also reaches a maximum of 4.26cm. The rooting effect of the regenerated shoot will be deteriorated by continuing to increase the concentration of DA-6.
TABLE 9 influence of different concentrations of DA-6 on rooting effect of shoot regeneration buds of Acanthopanax sieboldianus stem explant
| DA-6 concentration (mg/L) | Rooting percentage (%) | Average number of roots (strips) | Average root length (cm) |
| 0 | 46.79±2.11d | 2.64±0.14d | 2.78±0.12d |
| 0.15 | 55.73±3.29c | 3.57±0.12c | 3.22±0.17c |
| 0.3 | 78.85±3.38b | 4.50±0.19b | 3.66±0.27b |
| 0.6 | 90.23±1.32a | 6.08±0.23a | 4.26±0.23a |
| 1.2 | 73.36±4.50b | 4.28±0.32b | 3.34±0.19bc |
Note: data were analyzed for variance and Duncan multiple comparisons (P ≦ 0.05) using SPSS Statistics 17.0 statistical analysis software, with the difference in letters after the data indicating significant differences between treatments.
10. Influence of boron ions with different concentrations on rooting effect of regenerated shoot of explant of stem segment of cuirass
Taking MS solid culture medium containing 0.5mg/L TDZ and 2mg/L KT as basic culture medium, axially and horizontally inoculating the obtained stem segment explant on the basic culture medium containing 1.2mg/L nitroprusside for adventitious bud induction culture for 40 days, vertically inoculating the regenerated bud strip (adventitious bud) with length more than 1cm (with morphological lower end downwards) to the basic culture medium added with different concentrations of H 3 BO 3 Adventitious bud induction culture was performed on (MS solid medium +0.1mg/L IBA) rooting media (0, 0.2, 0.4, 0.8 and 1.6 mg/L) for 20 days, and it was found that 0.4mg/L H was added to the rooting basal medium 3 BO 3 The best rooting effect can be obtained.
Table 10 results show that H was added to the basal medium when rooting 3 BO 3 At a concentration of 0.4mg/L, the best raw material can be obtainedRoot effect, wherein the rooting rate reaches 57.31 percent at the maximum, the average root number also reaches 3.67 particles at the maximum, and the average root length also reaches 3.55cm at the maximum; however, the obtained adventitious roots are easily blackened, and leaves on the shoot are easily yellowed and even fall off. The concentration of boron ions is continuously increased, and the rooting effect of the regenerated bud sticks is deteriorated.
TABLE 10 influence of boron ions of different concentrations on the rooting effect of regenerated shoot of shoot explants of Trifolium ramosissimum
| H 3 BO 3 Concentration (mg/L) | Rooting percentage (%) | Average number of roots (strips) | Average root length (cm) |
| 0 | 46.06±3.59c | 2.62±0.14c | 2.75±0.14c |
| 0.2 | 51.78±1.62b | 3.24±0.22b | 3.15±0.19b |
| 0.4 | 57.31±2.75a | 3.67±0.15a | 3.55±0.15a |
| 0.8 | 47.39±2.76bc | 3.16±0.10b | 3.09±0.19b |
| 1.6 | 32.81±1.33d | 2.20±0.21d | 2.11±0.23d |
Note: data were analyzed for variance and Duncan multiple comparisons (P ≦ 0.05) using SPSS Statistics 17.0 statistical analysis software, with the difference in letters after the data indicating significant differences between treatments.
Claims (5)
1. A method for inducing adventitious bud regeneration of a stem explant of zoysia japonica is characterized by comprising the following steps:
a. taking the armored seeds, carrying out germination accelerating treatment and disinfection treatment, and then inoculating the seeds on an MS solid culture medium for aseptic germination culture; the MS solid culture medium consists of an MS culture medium, 30g/L sucrose and 8g/L agar,
pH 5.8-6.0；
b. taking a 15-25-day-grown gerbera ferruginea sterile plant, removing roots and leaves, and keeping stems; the stem is cut into small segments,
as a stem explant;
c. horizontally inoculating the stem section explant to an adventitious bud induction culture medium in the axial direction, and carrying out induction culture on the adventitious bud; the adventitious bud induction culture medium is any one of the following (1) to (3):
(1) Prepared from MS culture medium, TDZ 0.5mg/L, KT 2mg/L, nitroprusside 1.2mg/L, cuCl 2 0.3mg/L, 30g/L of cane sugar and 8g/L of agar, and the pH value is 5.8-6.0;
(2) Prepared from MS culture medium, TDZ 0.5mg/L, KT 2mg/L, sodium nitroprusside 1.2mg/L, mgSO 4 ·7H 2 0.5mg/L of O, 30g/L of cane sugar and 8g/L of agar, and the pH value is 5.8-6.0;
(3) Consists of MS culture medium, TDZ 0.5mg/L, KT 2mg/L, sodium nitroprusside 1.2mg/L, agCl 0.3mg/L, cane sugar 30g/L and agar 8g/L, and the pH value is 5.8-6.0;
d. inoculating the adventitious buds with the length of more than 1cm to a rooting culture medium, and inducing the adventitious buds to root to obtain a regenerated gerbera ferruginea plant; the rooting culture medium consists of MS culture medium, IBA 0.1mg/L, DA-6.6 mg/L, cane sugar 30g/L and agar 8g/L, and has pH of 5.8-6.0.
2. The method according to claim 1, wherein the pregermination treatment in the step a is as follows: soaking the seeds in 80 deg.C water for 10min.
3. The method of claim 1, wherein the sterilization treatment of step a is: sterilizing with 75% ethanol water solution for 30-45s, sterilizing with 2.5% NaClO water solution for 10-15min, shaking, and washing with sterilized water for 1min for 3-4 times.
4. The method of claim 1, wherein the cutting of the stem into small pieces in step b is cutting the stem into 0.5cm long pieces.
5. The method of claim 1, wherein the culturing conditions for the method for inducing regeneration of adventitious buds of a stem explant of Tripsammophyte are as follows: the illumination intensity is 2000lx, the illumination time is 12 hours/day, and the temperature is 25 +/-1 ℃.
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