CN117441617B - Tissue culture method of north shallot - Google Patents
Tissue culture method of north shallot Download PDFInfo
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Classifications
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/002—Culture media for tissue culture
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- Life Sciences & Earth Sciences (AREA)
- Developmental Biology & Embryology (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- Environmental Sciences (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention relates to the technical field of plant tissue culture, in particular to a tissue culture method of north shallot. The tissue culture method of the north shallot provided by the invention comprises the following steps: mature inflorescences of the shallot are used as explants, and the processes of inducing seed germination to form aseptic seedlings, proliferating the aseptic seedlings to form cluster buds and inducing adventitious buds to root are sequentially carried out. The tissue culture method of the welsh onion can remarkably improve the propagation efficiency of the welsh onion, reduce the limit of seasons on the production of the welsh onion, provide an effective method for the tissue culture and rapid propagation of the plant of the welsh onion, and facilitate the application of the welsh onion.
Description
Technical Field
The invention relates to the technical field of plant tissue culture, in particular to a tissue culture method of north shallot.
Background
North shallot (Allium schoenoprasum l.), alias: the herba Alii Fistulosi is perennial herb of Allium of Amaryllidaceae; the bulb is egg-shaped and cylindrical, and the diameter is 0.5-1 cm; the leaves are thin, pointed, long and cylindrical, hollow and slightly shorter than the scape; flower purple red or light red spherical shape with luster; the flower and fruit period is 7-9 months. North shallot is mainly distributed in the regions of the Uygur autonomous region of Xinjiang, altaishan mountain, europe, asian, zhongya, siberia, japan and North America, etc. The green onion can be used as a seasoning, and is scattered on vegetables after being cut into fine pieces to be used as an embellishment; but also can be used as gardening ornamental plants, can overwinter in most northern areas due to cold resistance, and avoids repeated planting due to perennial root plants. In addition, the welsh onion also has insect repellent performance, and can be used for preventing and controlling pests in gardens; a large amount of nectar can also be provided for pollinators. In the british plant investigation by the AgriLand project supported by the british insect pollinator initiative, north shallot was rated as the first 10 of most nectar yields (nectar per unit of coverage per year). The main propagation mode of the north shallot is sowing and separating, and the north shallot is planted in spring and autumn every year; at present, the report on plant tissue culture is not yet seen. The plant tissue culture technology has the advantages of short propagation period, high propagation coefficient, orderly and consistent emergence of seedlings and the like, and can provide a certain reference value for the preservation and propagation of excellent strains. Therefore, there is a need to develop a tissue culture method for north green onion.
Disclosure of Invention
The invention provides a tissue culture method of a north shallot.
Specifically, the invention provides the following technical scheme:
the invention provides a tissue culture method of a north shallot, which comprises the following steps: mature inflorescences of the shallot are used as explants, and the processes of inducing seed germination to form aseptic seedlings, proliferating the aseptic seedlings to form cluster buds and inducing adventitious buds to root are sequentially carried out.
Preferably, the mature inflorescence is an inflorescence of which the seeds are mature and wrapped by bracts.
The selection of a suitable stock plant part as an explant for tissue culture of the north shallot can directly influence the final result of the tissue culture of the north shallot. According to the invention, through a large number of attempts, the mature inflorescences of the north shallots (namely inflorescences with mature seeds and wrapped by bracts) are finally determined to be used as explants for tissue culture, and compared with the tissue culture by using bare seeds of the north shallots as the explants, the mature inflorescences not only can ensure better disinfection effect, but also have higher seed survival rate, and can effectively improve the induction success rate of the seeds, thereby remarkably improving the propagation efficiency of the tissue culture and reducing the cost of the tissue culture. According to the invention, the tissue culture of the north shallot is carried out by taking the mature inflorescence as the explant to reproduce the north shallot, so that the seed germination rate and the survival rate are effectively improved, the reproduction efficiency of the north shallot is remarkably improved, the steps of forming cluster buds by matching with the subsequent sterile seedling proliferation and inducing adventitious buds to take root can be better ensured, the proliferation coefficient and the rooting rate of the north shallot can be better ensured, and the rooting seedling is robust, so that the reproduction efficiency and the quality of the rooting seedling of the north shallot are remarkably improved.
Preferably, the mature inflorescence is derived from a parent plant which is robust and pest-free.
Preferably, the tissue culture method comprises the steps of female parent maintenance and explant selection, wherein in the female parent maintenance and explant selection, the test mother should have no plant diseases and insect pests, and the test mother should be healthy and reasonably maintained. Wherein, the yellow leaves are removed periodically in the reasonable maintenance of the female parent, so that sufficient sunlight is ensured, and the plant grows robustly.
The present invention further optimizes the media used for each step of tissue culture for the selection of the explants described above.
Specifically, in the tissue culture method, the culture medium for inducing the germination of seeds to form aseptic seedlings uses MS culture medium as basic culture medium and comprises the following components: 6-BA (6-benzylaminoadenine) 0.05-0.1mg/L, NAA (naphthylacetic acid) 0.05-0.1mg/L, sucrose 25-30g/L, agar 4-4.5g/L.
According to the invention, aiming at the mature inflorescence serving as an explant, in the stage of inducing seed germination to obtain aseptic seedlings, 6-BA and NAA with the concentrations are matched with the 6-BA and NAA serving as plant hormones, so that proliferation and expansion of cells can be remarkably promoted, and compared with the condition that an MS basic culture medium without hormone is added, other plant hormones or a combination thereof are used and other concentrations are used, the germination of the seeds can be more effectively promoted to form aseptic seedlings, the induction rate is improved, and the growth is more robust.
In the tissue culture method, the culture medium for forming the cluster buds by proliferation of the sterile seedlings takes MS culture medium as basic culture medium and comprises the following components: 6-BA 1.5-2mg/L, NAA 0-0.4mg/L, sucrose 25-30g/L, and agar 4-4.5g/L.
In the culture medium for forming cluster buds by the proliferation of the aseptic seedlings, the 6-BA with the concentration is singly used or the 6-BA with the concentration and NAA are matched to be used as plant hormones, so that the culture medium is more beneficial to inducing the proliferation of the aseptic seedlings to form the cluster buds, improving the proliferation coefficient and ensuring more efficient proliferation. When the addition amount of 6-BA is larger than the concentration, a small part of differentiated seedling plants obtained by final differentiation is malformed, so that the proliferation rate of the north shallot is lower.
In some embodiments of the invention, the medium used for propagation of the sterile seedlings to form the cluster buds is based on MS medium and comprises the following components: 6-BA 2mg/L, sucrose 25-30g/L, agar 4-4.5g/L.
In some embodiments of the invention, the medium used for propagation of the sterile seedlings to form the cluster buds is based on MS medium and comprises the following components: 6-BA 2mg/L, NAA 0.4mg/L, sucrose 25-30g/L, and agar 4-4.5g/L.
In the tissue culture method, the culture medium for inducing the adventitious bud to root takes 1/2MS culture medium as basic culture medium and comprises the following components: IBA (indolebutyric acid) 0.05-0.1mg/L, active Carbon (AC) 0.3-0.5g/L, sucrose 25-30g/L, and agar 4-4.5g/L.
IBA is added into a culture medium for inducing adventitious buds to root as a plant hormone, and the IBA can promote the adventitious buds of the shallot to root. The active carbon can adsorb the metabolite of the adventitious bud to enhance the permeability of the culture medium. Compared with a culture medium without IBA and active carbon, the rooting culture medium can root adventitious buds by 100%, and the rooting culture medium is short in time consumption, so that the propagation efficiency and survival rate of the green onions are improved.
Preferably, the pH of the above-described medium is 5.8-6.0.
In the tissue culture method, the culture temperature for inducing the germination of seeds to form aseptic seedlings and the proliferation of the aseptic seedlings to form cluster buds and the culture temperature for inducing the rooting of the adventitious buds is 23-25 ℃, the illumination intensity is 1500-1800Lux, and the illumination duration is 8-10h/d.
In the tissue culture method, the seeds are induced to germinate to form aseptic seedlings, and the seeds wrapped by the bracts are flatly inoculated on a culture medium.
And (3) in the cluster buds formed by the proliferation of the aseptic seedlings, cutting off the top ends of the germinated aseptic seedlings to a height of 1.5-2 cm, and vertically inoculating the aseptic seedlings in a culture medium.
In the induction of adventitious bud rooting, the proliferated adventitious buds are cut into single plants, and the single plants are vertically inoculated in a culture medium.
The tissue culture method further comprises the step of sterilizing the explant; the disinfection comprises: the explant is washed by water, and then is disinfected by 0.5-1% of benzalkonium bromide, 70-80% of alcohol and 0.5-1% of sodium hypochlorite in sequence.
Preferably, the benzalkonium bromide is sterilized by 1% benzalkonium bromide solution, the alcohol is sterilized by 75% alcohol solution, and the sodium hypochlorite is sterilized by 1% sodium hypochlorite solution.
Preferably, the explants are rinsed with sterile water after the sterilization of benzalkonium chloride, after the sterilization of alcohol, and after the sterilization of sodium hypochlorite.
Preferably, the time for sterilizing the benzalkonium bromide is 10-20min; the alcohol disinfection time is 1-2min; the time for sterilizing the sodium hypochlorite is 10-15min.
Further preferably, the time for sterilizing the benzalkonium bromide is 13-17min; the alcohol disinfection time is 1-1.5min; the time for sterilizing the sodium hypochlorite is 13-15min.
The use of the benzalkonium bromide for soaking and disinfection can reduce the surface tension of the bacteria to generate a certain cleaning effect, and in addition, the benzalkonium bromide can be adsorbed on the surface of the bacteria to change the permeability of cell membranes of the bacteria, so that enzymes, coenzyme and intermediate metabolites in the bacteria are discharged, the respiration and glycolysis processes of the bacteria are blocked, and the bacteria proteins are denatured to generate a bactericidal effect. The sterilization by alcohol immersion can kill a small amount of pathogens attached to the surface of the material, effectively eliminate the interference of pollution sources possibly attached to the surface of the explant, effectively increase the permeability of the sterilization agent to the surface of the explant material and further improve the success rate of the sterilization of the explant. Sodium hypochlorite belongs to a high-efficiency chlorine-containing disinfectant, and compared with mercury chloride, the sodium hypochlorite has less pollution to the environment and safer operation; sodium hypochlorite can form hypochlorous acid in water and act on bacterial proteins. Hypochlorous acid can act on cell walls, and has small molecules and no charge, so that the hypochlorous acid invades into cells to oxidize proteins or destroy phosphate dehydrogenase thereof, so that the cells die due to imbalance of sugar metabolism, and therefore, sodium hypochlorite can effectively kill pathogenic bacteria, and the success rate of disinfection of explants is further improved. In the sterilization of the explant, the novel Jieling is used for sterilization firstly, then the alcohol is used for sterilization, finally the sodium hypochlorite is used for sterilization, the novel Jieling and the alcohol have strong infiltration effects, the air on the surface of the explant can be discharged, and the infiltration of the disinfectant is facilitated, so that the penetrability of the disinfectant on the surface of the explant material is improved, the sterilization effect of the disinfectant is improved, and the success rate of the sterilization of the explant can be remarkably improved under the cooperation of the novel Jieling, the alcohol and the sodium hypochlorite. The sodium hypochlorite has a large killing effect on plants, if the soaking time is long, the growth of plant organs can be influenced and even killed, so that the soaking time and the use concentration are strictly controlled when the explant is disinfected by the sodium hypochlorite, and according to the experiment, the explant is soaked in a sodium hypochlorite solution with the concentration of 0.5-1% for the mature inflorescence of the green onion, and the time is controlled within the range of 10-15 min; when the sterilization time is longer, the pollution rate in the tissue culture process is lower, the sterilization effect is better, but the death rate of the explant is increased.
In some embodiments of the invention, the harvested explants are first rinsed with running water for 30 minutes and placed on an ultra clean bench; sterilizing with 1% benzalkonium bromide for 15min, and washing with sterile water for 4-5 times; sterilizing with 75% alcohol for 1.5min, and washing with sterile water for 4-5 times; finally, sterilizing with 1% sodium hypochlorite for 15 minutes, and rinsing with sterile water for 4-5 times.
The whole flow of the tissue culture method of the invention is as follows: sterilizing mature inflorescences on a healthy and plant-pest-free north onion mother plant, and then inducing the mature inflorescences by using a culture medium for inducing seed germination to form aseptic seedlings, wherein the mature inflorescences are used as seeds for germination of explants under the combined action of the culture medium for inducing seed germination to form aseptic seedlings and a proper culture environment to form aseptic seedlings; further utilizing the combined action of the culture medium for inducing proliferation and a proper culture environment to induce the proliferation of the single sterile seedling to form cluster buds; the adventitious buds are cut into single plants, the single plants are induced by utilizing a culture medium for inducing the adventitious buds to root, and the single plants form rooted seedlings under the combined action of the culture medium for inducing the adventitious buds to root and a proper culture environment.
The beneficial effects of the invention at least comprise: the tissue culture method of the north onion can remarkably improve the propagation efficiency of the north onion, reduce the limit of seasons on the production of the north onion, provide an effective method for the current blank north onion plant tissue culture field and facilitate the application of the north onion.
Drawings
In order to more clearly illustrate the invention or the technical solutions of the prior art, the following description will briefly explain the drawings used in the embodiments or the description of the prior art, and it is obvious that the drawings in the following description are some embodiments of the invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic diagram showing the induction of seed germination to form aseptic seedlings in the tissue culture process of North Allium fistulosum in Experimental example 1 according to the present invention by using the tissue culture method of example 1.
FIG. 2 is a schematic diagram showing the formation of multiple shoots by propagation of sterile seedlings during tissue culture of E.fistulosa in Experimental example 1 according to the present invention by the tissue culture method of example 1.
FIG. 3 is a schematic diagram showing rooting of adventitious buds during the tissue culture of Allium fistulosum in Experimental example 1 according to the present invention by using the tissue culture method of example 1.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings, and it is apparent that the described embodiments are some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The benzalkonium bromide used in the following examples was a solution having a concentration of benzalkonium bromide as an active ingredient of 27g/L to 33g/L, and was purchased from Shandong Li Erkang medical technologies Co., ltd (500 mL specification); the alcohol is European clean 75% sterile alcohol purchased from European Tuber Biotechnology Co., ltd (500 mL specification); sodium hypochlorite is a solution with an available chlorine content of 10.0% and is available from Tianjin chemical company, inc. (500 mL specification).
The north onion stock plant used in the following examples was from all companies of Beijing flower country flower science and technology research.
The tissue culture method of the north shallot provided by the invention specifically comprises the following steps:
1. female parent maintenance and explant selection:
(1) And (3) curing female parent: isolating the female parent from other varieties, placing the female parent in a separate area in a greenhouse for culturing for 30-60 days, timely removing dead leaves in the growth process, ensuring sufficient sunlight and enabling plants to grow robustly;
(2) Selection of explants: and selecting a parent plant which grows well and has good characters and no plant diseases and insect pests from the female parent, and taking an inflorescence which is mature in seeds on the parent plant and is wrapped by bracts as an explant.
2. Cleaning and disinfection of explants:
(1) Flushing: washing the collected explant with running water for 30 minutes, washing off surface dust, and then putting the washed explant into an ultra-clean workbench for airing;
(2) Sterilizing with benzalkonium bromide: the explant is cut into individual florets after being dried, the pedicel is reserved about 0.5 cm, 1% of benzalkonium bromide is used for vibration disinfection for 15 minutes, and then the explant is rinsed with sterile water for 4-5 times for 1-2 minutes each time;
(3) Alcohol sterilization: then shake sterilizing with 75% alcohol for 1.5min, and washing with sterile water for 4-5 times, each for 1-2min;
(4) Sterilizing with sodium hypochlorite: finally, 1% sodium hypochlorite is used for vibrating and sterilizing for 10-15 minutes, sterile water is used for rinsing for 4-5 times, each time is 1-2 minutes, and the water is slightly dried, so that the sterilized explant is obtained.
3. Inducing germination of seeds to form aseptic seedlings: spreading and inoculating the seeds wrapped by the sterilized bracts to a culture medium for inducing the seeds to germinate, and inducing the seeds to germinate to form aseptic seedlings; the formula of the culture medium for inducing seed germination is as follows: MS culture medium, adding 6-BA 0.05-0.1mg/L, NAA 0.05-0.1mg/L, sucrose 25-30g/L, agar 4-4.5g/L, pH 5.8-6.0; the culture temperature is 23-25 ℃, the illumination intensity is 1500-1800Lux, and the illumination duration is 8-10h/d.
4. Sterile seedlings proliferate to form clumps of buds: removing basal seed shells of the obtained aseptic seedlings, cutting off top ends of the aseptic seedlings to a height of 1.5-2 cm, and vertically inoculating the aseptic seedlings to a culture medium for inducing the proliferation of the aseptic seedlings; the formula of the culture medium for inducing the proliferation of the aseptic seedlings is as follows: MS culture medium, 6-BA 2mg/L, NAA 0-0.4mg/L, sucrose 25-30g/L, agar 4-4.5g/L and pH 5.8-6.0; the culture temperature is 23-25 ℃, the illumination intensity is 1500-1800Lux, and the illumination duration is 8-10h/d.
5. Induction of adventitious bud rooting: cutting the adventitious buds obtained by multiplication into independent adventitious buds, and inoculating the independent adventitious buds to a culture medium for inducing the adventitious buds to root; the formula of the culture medium for inducing adventitious bud to root is as follows: 1/2MS culture medium, IBA 0.05-0.1mg/L, active carbon 0.3-0.5g/L, sucrose 25-30g/L, agar 4-4.5g/L, pH 5.8-6.0; the culture temperature is 23-25 ℃, the illumination intensity is 1500-1800Lux, and the illumination duration is 8-10h/d.
The present invention will be described in further detail with reference to the accompanying drawings, examples and comparative examples and their related experimental results.
Example 1
The embodiment provides a tissue culture method of north shallot, comprising the following steps:
1. female parent maintenance and explant selection:
(1) And (3) curing female parent: isolating the female parent from other varieties, placing the female parent in a separate area in a greenhouse for culturing for 30-60 days, timely removing dead leaves in the growth process, ensuring sufficient sunlight and enabling plants to grow robustly;
(2) Selection of explants: and selecting a mother plant which grows well and has good characters and no plant diseases and insect pests from the female parent, and taking an inflorescence which is mature in seeds on the mother plant and is wrapped by bracts.
2. Cleaning and disinfection of explants:
(1) Flushing: washing the collected explant with running water for 30 minutes, washing off surface dust, and then putting the washed explant into an ultra-clean workbench for airing;
(2) Sterilizing with benzalkonium bromide: cutting the explant into individual flowers after airing, reserving pedicel of about 0.5 cm, vibrating and sterilizing for 15 minutes by using 1% benzalkonium bromide, and then rinsing for 4-5 times by using sterile water for 1-2 minutes each time;
(3) Alcohol sterilization: then shake sterilizing with 75% alcohol for 1.5min, and washing with sterile water for 4-5 times, each for 1-2min;
(4) Sterilizing with sodium hypochlorite: finally, 1% sodium hypochlorite is used for vibrating and sterilizing for 15 minutes, sterile water is used for rinsing for 4-5 times, each time is 1-2 minutes, and the water is slightly dried, so that the sterilized explant is obtained.
3. Inducing germination of seeds to form aseptic seedlings: cutting off redundant parts of the sterilized explant, only reserving seeds with outer skins, peeling off inflorescence outer skins, flatly inoculating the seeds wrapped by the bracts to a culture medium for inducing seed germination, and inducing the seeds to germinate to form aseptic seedlings; the formula of the culture medium for inducing seed germination is as follows: MS culture medium, adding 0.1mg/L of 6-BA, 0.1mg/L of NAA, 30g/L of sucrose, 4.5g/L of agar and pH 6.0; the culture temperature is 23 ℃, the illumination intensity is 1800Lux, and the illumination time is 10h/d.
4. Sterile seedlings proliferate to form clumps of buds: removing basal seed shells of the obtained aseptic seedlings, cutting off top ends of the aseptic seedlings to a height of 1.5-2 cm, and vertically inoculating the aseptic seedlings to a culture medium for inducing the proliferation of the aseptic seedlings; the formula of the culture medium for inducing the proliferation of the aseptic seedlings is as follows: MS culture medium, adding 2mg/L of 6-BA, 30g/L of sucrose, 4.5g/L of agar and pH 6.0; the culture temperature is 23 ℃, the illumination intensity is 1800Lux, and the illumination time is 10h/d.
5. Induction of adventitious bud rooting: cutting the adventitious buds obtained by multiplication into independent adventitious buds, and inoculating the independent adventitious buds to a culture medium for inducing the adventitious buds to root; the formula of the culture medium for inducing adventitious bud to root is as follows: 1/2MS culture medium, adding IBA 0.1mg/L, active carbon 0.3g/L, sucrose 30g/L, agar 4.5g/L, and pH 6.0; the culture temperature is 23 ℃, the illumination intensity is 1800Lux, and the illumination time is 10h/d.
The conditions parameters and the medium formulations used in the above steps are shown in Table 1.
Table 1 parameters and media formulations used for each step
Example 2
This example provides a tissue culture method of Allium fistulosum, which is identical to the procedure of example 1, except that the condition parameters and the medium formulation used for part of the procedure are as shown in Table 1. The procedure of example 2 was identical to that of example 1, except for the condition parameters and the medium listed in Table 1.
Comparative example 1
This comparative example provides a tissue culture method of north shallot, which differs from the method of example 1 only in that: no hormone is added to the medium which induces the seeds to germinate to form aseptic seedlings.
Comparative example 2
This comparative example provides a tissue culture method of north shallot, which differs from the method of example 1 only in that: in the culture medium for inducing seed germination to form aseptic seedlings, the concentration of 6-BA is 2mg/L, and the concentration of NAA is 0.1mg/L.
Comparative example 3
This comparative example provides a tissue culture method of north shallot, which differs from the method of example 1 only in that: the sterilization time was 20min using sodium hypochlorite, and the concentration of 6-BA in the induced sterile seedling proliferation medium was adjusted to 3mg/L.
Comparative example 4
This comparative example provides a tissue culture method of north shallot, which differs from the method of example 1 only in that: IBA is not added into the rooting culture medium.
Comparative example 5
This comparative example provides a tissue culture method of north shallot, which differs from the method of example 1 only in that: the explants were selected from naked mature seeds.
Experimental example 1
The tissue culture method of each example and comparative example was used to perform tissue culture on the north shallot, and the investigation parameters of the cultures at each stage in the corresponding culture process were recorded, and the pollution rate and death rate of the north shallot were calculated, and the specific investigation parameters and recording results are shown in table 2.
Meanwhile, the disinfection effect of the explant and the quality of adventitious buds formed in the cultivation process of the shallot were recorded, and the specific recording results are shown in Table 3.
In the process of performing the tissue culture of the shallot by using the tissue culture method of the embodiment 1, a schematic diagram of inducing the seed to germinate and form the aseptic seedling is shown in fig. 1, a schematic diagram of proliferating and forming the cluster bud is shown in fig. 2, and a schematic diagram of rooting the adventitious bud is shown in fig. 3. Since in the implementation process, each five tissue culture flasks were grouped into one group, and each flask was 20 seeds, the above-mentioned fig. 1 does not represent the overall germination level, and the specific result data are shown in tables 2 and 3.
TABLE 2 tissue culture results of North Allium (1)
TABLE 3 tissue culture results of North Allium (2)
By combining the tissue culture methods and the tissue culture results of the embodiments and the comparative examples, the invention selects the inflorescence with mature seeds but wrapped bracts on the mother plant of the welsh onion as the explant, and selects the proper culture medium formula and culture conditions for the tissue culture of the welsh onion in each tissue culture stage, thereby not only improving the disinfection success rate of the explant of the welsh onion, but also improving the survival rate and the induction rate of the seeds, improving the proliferation coefficient of adventitious buds, simultaneously, having better rooting effect of the welsh onion, further improving the proliferation efficiency of the welsh onion, reducing the limitation of seasons on the production of the variety, and further promoting the application of the welsh onion.
Specifically, first, selecting an appropriate stock plant site as an explant for tissue culture of north shallot can directly affect the final result of tissue culture of north shallot. The results of the cultures of comparative example 1 and comparative example 5, under the same tissue culture conditions and no pest requirements, the explant selected in example 1 was mature seed-but bract-wrapped inflorescence on the stock plant, while the explant selected in comparative example 5 was naked mature seed. As can be seen from the results of tables 2 and 3, the contamination rate obtained by the tissue culture method of example 1 was 5%, the death rate was 0%, the seed germination induction rate was 80%, the induction rate was high, and a large amount of aseptic seedlings were obtained by induction; the contamination rate obtained by the tissue culture method of comparative example 5 was 10%, the mortality rate was 15%, the contamination rate and the mortality rate were significantly higher than those of example 1, the induction rate of seed germination was 70%, and significantly lower than that of example 1.
Therefore, the effect of the selection of the explant on the plant tissue culture is greatly influenced, and the plant tissue culture is performed by selecting the proper parent plant part as the explant, so that the effect of the north onion tissue culture can be improved. According to the invention, the inflorescence with mature seeds and wrapped by bracts on the stock plant is selected as the explant for tissue culture of the north shallot, so that the pollution rate and the death rate are effectively reduced, and the germination rate of the north shallot is improved.
Second, selecting the proper time for sterilization with sodium hypochlorite also affects the tissue culture results of the north shallot. The disinfection results of comparative example 1 and comparative example 3. The contamination rate and mortality rate of example 1 were 5% and 0%, respectively, and the explant expression after sterilization was normal. The contamination rate and mortality rate of comparative example 3 were 2% and 30%, respectively, and the explants appeared to be partially brown. Although comparative example 3 has a lower contamination rate than example 1, it has a high mortality rate. Therefore, the proper sodium hypochlorite sterilization time has a great influence on the tissue culture result of the chives, and the survival rate of the chives explants can be improved by selecting the proper sodium hypochlorite sterilization time. The method selects 10-15min as the disinfection time of the sodium hypochlorite, and effectively improves the survival rate of the north onion explants.
In addition, the culture medium formula and the culture conditions selected at each stage of the tissue culture of the north green onion can also have an influence on the result of the tissue culture of the north green onion.
Wherein, in the step of inducing seed germination to form aseptic seedlings, the culture results of the example 1 and the comparative examples 1 and 2 show that the induction rate of the seed germination of the example 1 is 80 percent and the seed germination rate is higher; whereas the seed germination of comparative example 1 had an induction rate of only 50%, the germination rate was low, and the seedlings were thin and weak. Whereas comparative example 2 had a seed germination induction of 75%, which was lower than example 1, and had vitrification, and the seed germination quality was poor. Thus, the addition of a lower concentration of the combination of 6-BA and NAA during seed germination facilitates seed germination.
In the step of inducing proliferation of aseptic seedlings to form cluster buds, the results of the culture of example 1 and comparative example 3 were compared, the phytohormone used in the induction of proliferation of aseptic seedlings to form cluster buds in example 1 was a relatively low concentration of 6-BA, and the phytohormone used in the induction of proliferation of aseptic seedlings to form cluster buds in comparative example 3 was a relatively high concentration of 6-BA. As can be seen from the results in tables 2 and 3, the proliferation coefficient of the cluster buds obtained by the tissue culture method of example 1 is 3-4 times, a large number of adventitious buds are obtained by induction, the growth speed is high, the plants are relatively strong, and the proliferation rate is high; the proliferation coefficient of the cluster buds obtained by the tissue culture method of the comparative example 3 is 2-3, and the deformity of a small part of the differentiated seedling plants of the comparative example 3 occurs, so that the proliferation rate is low.
Therefore, the selection of plant hormone in the step of inducing the sterile seedlings to proliferate to form the cluster buds has a great influence on the tissue culture effect, and the invention selects 6-BA with lower concentration or the combination of 6-BA and NAA as the plant hormone of the culture medium used in the step of inducing the sterile seedlings to proliferate to form the cluster buds, so that the quality of adventitious buds induced in the tissue culture of the north shallot can be improved, a large number of adventitious buds can be induced, the induction rate and proliferation coefficient of the north shallot can be improved, and the induction time of the formation of the adventitious buds of the north shallot can be shortened. Thus, in the step of inducing the explant to form adventitious buds, a relatively low concentration of 6-BA or a combination of 6-BA and NAA increases the reproductive efficiency of North Allium, whereas a relatively high concentration of 6-BA instead inhibits the proliferation of clumped buds.
Finally, the addition of IBA in the culture medium for inducing adventitious buds to root has a great promoting effect on rooting. As can be seen from comparative examples 1 and 4, example 1, in which an appropriate amount of IBA was added, had a rooting rate of 100%, and had a rapid rooting and a good rooting effect. The rooting rate of comparative example 4 without IBA is only 80%, and the rooting rate is low and weak.
In conclusion, the tissue culture method of the north shallot provided by the invention has the advantages that the explant selects the inflorescence with mature seeds and wrapped bracts on the mother plant, the survival rate and germination rate of the seeds are high, the sterile seedlings grow strongly, the proliferation coefficient is high, the growth speed is high, and the adventitious buds grow strongly; meanwhile, the rooting effect of the north shallot is good, the rooting rate reaches 100%, the propagation efficiency and the quality of rooting seedlings of the north shallot are effectively improved, the limitation of seasons on the production of the variety is reduced, the application of the north shallot is facilitated, and the blank of the north shallot in the field of plant tissue culture is filled.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims (6)
1. A method of tissue culture of allium fistulosum, the method comprising: taking a mature inflorescence of the shallot as an explant, sequentially performing induction seed germination to form aseptic seedlings, proliferation of the aseptic seedlings to form cluster buds and induction of adventitious buds to root;
the mature inflorescence is an inflorescence of which the seeds are mature and wrapped by bracts;
the culture medium for inducing seed germination to form aseptic seedlings takes MS culture medium as basic culture medium and comprises the following components: 6-BA 0.05-0.1mg/L, NAA 0.05-0.1mg/L, sucrose 25-30g/L, agar 4-4.5g/L;
the culture medium for forming cluster buds by proliferation of the sterile seedlings takes MS culture medium as basic culture medium and comprises the following components: 1.5-2mg/L of 6-BA, 0-0.4mg/L of NAA, 25-30g/L of sucrose and 4-4.5g/L of agar;
the culture medium for inducing adventitious bud rooting takes 1/2MS culture medium as basic culture medium and comprises the following components: IBA 0.05-0.1mg/L, active carbon 0.3-0.5g/L, sucrose 25-30g/L, agar 4-4.5g/L.
2. The tissue culture method of north herba Alii Fistulosi according to claim 1, wherein the pH of the culture medium is 5.8-6.0.
3. The tissue culture method of north shallot according to claim 1 or 2, characterized in that the culture temperature of inducing seed germination to form aseptic seedling, aseptic seedling proliferation to form cluster bud and adventitious bud rooting is 23-25 ℃, the illumination intensity is 1500-1800Lux, and the illumination duration is 8-10h/d.
4. The tissue culture method of north shallot according to claim 1 or 2, characterized in that said inducing seed germination forms aseptic seedlings, seed wrapped with bracts is spread and inoculated on a culture medium;
and/or, the aseptic seedlings are proliferated to form cluster buds, the top ends of the germinated aseptic seedlings are cut off to be 1.5-2 cm high, and the aseptic seedlings are vertically inoculated in a culture medium;
and/or, in the induction of adventitious bud rooting, cutting the proliferated adventitious buds into single plants, and vertically inoculating the single plants into a culture medium.
5. A method of tissue culture of allium fistulosum as claimed in claim 1 or claim 2, wherein the method further comprises the step of sterilizing the explant;
the disinfection comprises: the explant is washed by water, and then is disinfected by 0.5-1% of benzalkonium bromide, 70-80% of alcohol and 0.5-1% of sodium hypochlorite in sequence.
6. The tissue culture method of north herba Alii Fistulosi of claim 5, wherein the time of sterilizing the new Jieli is 10-20min; the alcohol disinfection time is 1-2min; the time for sterilizing the sodium hypochlorite is 10-15min.
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| WO2000016610A1 (en) * | 1998-09-24 | 2000-03-30 | Univerza V Ljubljani, Biotehniška Fakulteta | A process for the induction of direct in vitro organogenesis in onion |
| CN101960991A (en) * | 2010-11-15 | 2011-02-02 | 南京农业大学 | Onion callus induction method and special culture medium thereof |
| CN106035080A (en) * | 2016-05-30 | 2016-10-26 | 甘肃省治沙研究所 | Allium mongolicum regenerated seedling and allium mongolicum callus acquisition method |
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