CN110547197A - Method for tissue culture and rapid propagation of seedlings through air pineapple lateral buds - Google Patents
Method for tissue culture and rapid propagation of seedlings through air pineapple lateral buds Download PDFInfo
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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
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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
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Abstract
The invention discloses a method for tissue culture and rapid propagation of seedlings by using lateral buds of tillandsia undulata, which comprises the following steps: 1) collecting lateral buds of the tillandsia fruits; 2) sterilizing and culturing lateral buds; 3) induction culture of cluster buds; 4) carrying out rapid propagation and differentiation culture on the bud seedlings; 5) strong seedling and rooting culture; 7) hardening and transplanting aseptic seedlings. The method has the advantages of simple operation, convenient material acquisition, obviously higher propagation coefficient than outdoor, high seedling growth speed and the like, the lateral buds can be propagated to about 11 buds on average about 120 days by starting rapid propagation culture, not only the propagation coefficient is obviously higher than outdoor, but also the seedling growth speed is high and robust, a large number of tissue culture seedlings with consistent characters and growth consistency and robustness can be rapidly propagated in a short time, the purpose of rapid propagation is achieved, the tissue culture seedlings are easy to survive by moving the tissue culture seedlings into the outdoor through seedling exercising, and the survival rate is as high as 100%.
Description
Technical Field
The invention relates to the field of plant breeding, in particular to a method for tissue culture and rapid propagation of seedlings through air pineapple lateral buds.
Background
The air plants are various in types, different in plant shapes, various in posture, colorful in flower and leaf and beautiful in personality, the air plants are not only high in ornamental value and good in decorative effect, but also have strong air purification functions of absorbing formaldehyde, phenylenes, CO 2 and the like, and are more and more popular as ornamental plants, the potential commercial opportunities are unavailable due to the characteristics.
At present, the main reason for restricting the development and popularization of the air pineapples is that seedlings are few and expensive, especially some rare provenances. The expanded propagation of the tillandsia seedling generally adopts mature seed propagation or plant division propagation, most varieties in seed propagation generally need 2-6 months after pollination is successful until seed pods can be seen, and the seed pods need 3-30 months after maturation according to the varieties; whether seeds germinate and seedlings can grow normally is easily influenced by environmental changes such as temperature, humidity, ventilation and the like, seedlings are very fragile in the initial stage and grow very slowly, the seedlings within 2 years are usually required to be transplanted randomly and sprayed with any chemical substances and fertilizers, and otherwise the seedlings are extremely easy to die; the management levels of different climatic humidity requirements of different regions are different, and the adopted matrixes are also different; in a word, the air pineapple seeds have high requirements on the propagation production difficulty, and have the problems that the characters of hybrid progeny are separated, the excellent characters of female parents are difficult to maintain, the uniformity of the progeny groups is poor, the quality of improved seeds is reduced and the like; therefore, the expanded propagation of the air plants in the current production mainly adopts the mode of lateral bud plant division propagation; however, the lateral bud usually grows a sub-plant on one side of the mother plant after the flower blooms, the number of the sub-plants is small, the sub-plants can be separated from the mother plant when the sub-plants grow to 1/3-1/2 of the mother plant, and therefore the propagation coefficient of the branch plant is also extremely low; the recent rise of plant tissue culture has provided an effective way for the propagation of tillandsia (Dingkui et al, 2010). However, the current research reports on the aspect of tissue culture and rapid propagation of the tillandsia are very few, and particularly, the tissue culture and rapid propagation by utilizing lateral buds of the tillandsia is not seen yet. The plum compares the growth conditions of the seeds of the Spodoptera specie with three different nutrient components on MS (or agar only) culture media and by adding certain hormone (auxin or cytokinin) in the MS culture media, and the results show that the seeds can grow seedlings on 3 culture media, but the MS culture media enable the seedlings to grow fastest; furthermore, the seedlings added with auxin (NAA and IBA) in the MS culture medium grow obviously faster than those without the auxin, and the number of leaves is more; cytokinin (6-BA) growth regulators are added into the MS culture medium, so that the growth of the seedlings is not promoted. The invention discloses an air pineapple tissue culture rapid propagation method disclosed by Lepeng, which is mainly characterized in that 4-5 classes of seedlings can be separated from germination of one seed to final separation of the one seed through sterile seed germination and rapid propagation. It can be seen that the above reports are different from the present invention in terms of the method technology, the formula ratio of the culture medium, etc., and the experimental results and effects are also different (or completely). The extremely low propagation coefficient is always a main factor for preventing the commercial production and the industrial development of the air pineapple, particularly the excellent and rare germplasm resources.
disclosure of Invention
The purpose of the invention is as follows: the invention provides a method for tissue culture and rapid propagation of seedlings by air pineapple lateral buds, aiming at the defects of the prior art of the air pineapple.
The technical scheme is as follows: in order to solve the technical problems, the invention provides a method for tissue culture and rapid propagation of seedlings by air pineapple lateral buds, which comprises the following steps:
1) collecting lateral buds of the air plants: the size of the lateral bud is preferably 1-5cm, and the cut explant is taken back to a laboratory for disinfection treatment as soon as possible;
2) Disinfection and sterilization of lateral buds and start culture: the disinfectant used for lateral bud disinfection and sterilization comprises 1% of sodium hypochlorite and 2-3 drops of Tween mixed solution, 70-75% of alcohol and 0.1% of mercuric chloride; inoculating the sterilized lateral buds to a starting culture medium, starting culture in a shading mode for about 15-28d, counting the infection rate of the lateral buds, and observing the starting culture of the lateral buds and the growth and development conditions of lateral bud embryonic calluses;
3) Induced differentiation culture of cluster buds: transferring the sterile side bud explant (the side bud embryonic callus) which is started to be cultured in the step 2) to a cluster bud induced differentiation culture medium, culturing under 1500-2000LX light, and observing the growth and development conditions of the cluster buds;
4) and (3) rapid propagation and multiplication culture of the bud seedlings: cutting the cluster buds obtained in the step 3) into 2-3 buds, transferring the buds to a bud seedling rapid propagation culture medium, culturing under 1500-2000LX light, and observing and counting bud proliferation times and bud seedling growth and development conditions after 28-30 days;
5) strong seedling and rooting culture: transferring the cluster bud seedling cut into 2-3 plants into a strong seedling rooting culture medium, and culturing under 2000-2500LX light;
7) Hardening and transplanting aseptic seedlings.
Wherein the starting culture medium in the step 2) is MS +6-BA1-3mg/L + NAA1-2mg/L +2, 4-D0.5mg/L.
Wherein the cluster bud induction culture medium in the step 3) is MS +6-BA 1-2mg/L + NAA 0.2-0.5 mg/L.
wherein the culture medium for rapid propagation and proliferation of the bud seedlings in the step 4) is MS +6-BA2.0mg/L + NAA0.2mg/L.
Wherein the strong seedling rooting culture medium in the step 5) is Ms +6-BA 0.1-0.5 mg/L + IBA 0.2-0.5 mg/L.
most preferably, the best starting medium formula of the lateral bud of the invention is MS +6-BA2.0mg/L + NAA2.0mg/L +2, 4-D0.5mg/L; the best formula of the cluster bud induction culture medium is MS +6-BA1.0mg/L + NAA0.5mg/L; the best formula of the bud rapid propagation and proliferation medium is MS +6-BA2.0mg/L + NAA0.2mg/L.
Has the advantages that: compared with the prior art, the invention has the following advantages: the method has the advantages of simple operation, convenient material acquisition, obviously higher propagation coefficient than outdoor, high seedling growth speed and the like, the lateral buds can be propagated to about 11 buds on average about 120 days by starting rapid propagation culture, not only the propagation coefficient is obviously higher than outdoor, but also the seedling growth speed is high and robust, a large number of tissue culture seedlings with consistent characters and consistent growth and robustness can be rapidly propagated in a short time, the purpose of rapid propagation is completed, therefore, a large number of tissue culture seedlings with consistent characters and consistent growth and robustness can be rapidly propagated in a short time, the tissue culture seedlings are easy to survive after being transplanted to outdoor through seedling exercising, and the survival rate is as high as 100%. Therefore, the technology not only solves the problems of difficult seed propagation, low propagation coefficient by plant, slow growth and the like of the air plants, but also is beneficial to large-scale industrialized production of excellent rare germplasm, and provides an effective way for commercial production and industrial development of the air plants.
drawings
FIG. 1 Disinfection of lateral buds;
FIG. 2 lateral bud embryogenic callus induction;
FIG. 3 is a side bud multiple bud propagation culture;
FIG. 4 rooting culture of strong seedlings;
FIG. 5 shows the disinfection and the start of the culture of different varieties of lateral bud explants;
FIG. 6 induction of lateral Harris bud embryogenic callus and differentiation and rapid propagation of multiple shoots with proliferation;
FIG. 7 shows the induction of lateral bud embryogenic callus and the differentiation and rapid propagation of multiple shoots;
FIG. 8 is the induction of lateral bud embryogenic callus of genipin and the differentiation and rapid propagation and proliferation culture of multiple shoots.
the specific implementation mode is as follows:
The invention is further illustrated by the following examples.
All the culture temperatures in the examples of the present invention were 20 to 28 ℃ and, although not specifically indicated, the culture medium pH was 5.8, sucrose was 30g/L, and agar was 6.5 g/L. The variety of the air pineapple is 18 varieties such as Harris, Dasanchi, eidolon and the like, and is shown in figure 5.
Example 1: selection and disinfection of air pineapple lateral buds
1. Selecting lateral buds: considering the particularity of the growth of the lateral buds of the air pineapples, such as slow growth speed, weak growth capacity, crisp and tender leaves and stems, a small amount of accumulated water at the top ends of the buds of some silver leaf varieties and the like, the difficulty of collecting and disinfecting the lateral bud explants is increased; much detail needs to be taken in advance during the collection and sterilization of the air pineapple side-bud explants. Generally, the proper material taking time is better when the temperature does not rise again in early spring or the air turns to cool in winter in the end of autumn and the beginning, if the explant is collected in the high-temperature and high-humidity weather condition in midsummer, the explant which grows well and has no any disease to the stock plant is collected in the weather of continuous sunny for at least 3 days. In order to ensure high survival rate, the side bud is preferably selected to be 2-5cm high, a knife for cutting the side bud needs high-temperature disinfection and sterilization in advance, the knife edge is sharp so as to prevent the cut from being susceptible to sawtooth infection, and the cut explant is brought back to a laboratory for disinfection treatment as soon as possible.
2. Disinfection and sterilization of lateral buds and start culture: as shown in figure 5, soaking lateral bud in washing powder for 12-20min, and washing in running water for 30-50 min. And then 1% sodium hypochlorite and 2-3 drops of Tween are prepared into a mixed disinfectant to be disinfected for 8-10min, the disinfectant is washed by sterilized water for 3 times, surface water is absorbed and dried, the disinfectant is taken onto a super-clean workbench, the disinfectant is firstly soaked in 70-75% alcohol for 30s, then the disinfectant is put into 0.1% mercuric chloride to be disinfected for 8-15min, and the disinfectant is washed by sterile water for 8-10 times, wherein the disinfectant cannot be shaken violently during the washing. Wiping water with a sterilized absorbent paper, particularly wiping off accumulated water, and hanging the lateral buds upside down for 30s with tweezers to ensure that no accumulated water exists in the lateral buds; then, the leaves are stripped layer by layer from outside to inside by using tweezers, only 2-3 leaves at the top are kept, a small amount of browned stems at the bottom are cut off by scissors, and the treated explants are inoculated to different starting culture media. In order to reduce cross infection, 1 strain is placed in each bottle, 20 bottles are inoculated together, shading starting culture is carried out for about 15-28d, the infection rate of lateral buds is counted, and the starting culture of the lateral buds and the growth and development conditions of lateral bud embryonic calluses are observed; the results are shown in Table 1 (in the case of the large trichrome). The lateral bud infection rate is 100 percent of lateral bud number of infected bacteria/lateral bud number of test
TABLE 1 Effect of different starting Medium formulations on lateral bud infection rate and germination growth development
As can be seen from table 1: all the lateral buds in the formula have a certain bacterial contamination rate, but along with the increase of the concentration of auxin, the callus at the base part of the lateral buds gradually becomes larger, and the bacterial contamination rate gradually decreases; the formulation C4 and C5 have the largest callus degree, but the embryogenic callus growth and development of the formulation C5 is obviously better than that of other formulations, the contamination condition of other varieties, the embryogenic callus degree of lateral buds and the trichromatic flower are similar, so that the larger the callus growth is, the better the growth is, and which formulation is more favorable for the induction and bud proliferation of the cluster buds, and the materials are transferred to the following induction and differentiation of the cluster buds and the rapid propagation culture of the buds to be continuously observed.
Example 2: induced differentiation of cluster buds and rapid propagation and multiplication culture of bud seedlings
1. Induced differentiation culture of cluster buds: the above-mentioned sterile side shoot explants (side shoot embryogenic callus) were transferred to the following cluster shoot differentiation induction medium to induce the generation of cluster shoots, and cultured under 1500-2000LX light for 56d to observe the growth and development of cluster shoots (see FIG. 6, FIG. 7, FIG. 8 and Table 2).
TABLE 2 Effect of different cluster bud Induction Medium formulations on the growth and development of cluster buds
As can be seen from table 2: except that C01 and C02 are controls, no cluster buds are generated under the influence of the starting medium formula, and all serial numbers (all formulas) can induce cluster buds, but the cluster bud generation degree is greatly different, wherein the best serial number is C51, namely MS +6-BA1.0+ NAA0.5 cluster bud induction medium formula, when an explant (embryogenic callus) with the serial number C5 is transferred onto the cluster bud induction medium C51, the cluster bud generation degree is the most, and the best starting medium formula is C5 serial number and the best cluster bud induction medium formula is C51 serial number according to the analysis.
2. And (3) rapid propagation and multiplication culture of the bud seedlings: and (3) transferring the sterile cluster buds (more of which need to be cut into 2-3 buds) to a bud seedling rapid propagation culture medium, performing rapid propagation culture under 1500-2000LX light, observing the growth and development conditions of the bud seedlings, taking single lateral buds as a control, and counting the bud proliferation times after 28 days (see fig. 6, 7, 8 and table 3).
TABLE 3 Effect of different sprout stock multiplication medium formulations on sprout multiplication
as can be seen from table 3: except that the serial numbers C01 and C02 are generated by contrast non-bud seedlings, the materials of all the serial numbers grow well when being transferred into a bud seedling rapid propagation culture medium, but the bud propagation multiples are greatly different, and the best serial number C51 is obtained by statistics, namely the formula of the bud rapid propagation culture medium is MS +6-BA2.0+ NAA 0.2; analysis found that the degree of cluster bud formation was highest when C51 was cultured on the cluster bud induction medium; the formula of the starting culture medium with the best lateral bud obtained by the research is MS +6-BA2.0+ NAA2.0+2, 4-D0.5; the best formula of the cluster bud induction culture medium is MS +6-BA1.0+ NAA 0.5; the best formula of the bud rapid propagation and proliferation culture medium is MS +6-BA2.0+ NAA 0.2; by the rapid propagation culture starting, one lateral bud can be propagated to about 11 buds in about 120 days on average, the propagation coefficient is obviously higher than that of the outdoor side, the seedling growth speed is high, the seedlings are robust, a large number of tissue culture seedlings with consistent properties and growth consistency can be rapidly propagated in a short time, and the purpose of rapid propagation is achieved. The bud multiplication times of other varieties are similar to those of the three-color flower.
example 3: strong seedling rooting culture and transplantation of seedlings:
1. Strong seedling and rooting culture: the bud seedlings are divided into 2-3 plant clusters and transferred into a strong seedling or strong seedling rooting culture medium, referring to figure 4, the bud seedlings are cultured for 14h/d under 2000-2500LX light, and about 28 days, the small bud seedlings can grow into normal and strong plants with a plurality of roots, stems and leaves in 100 percent. Directly transferring the strong seedling with better growth condition into a strong seedling culture medium for culture according to the condition of the bud seedling, firstly culturing the weak seedling in a strong seedling rooting culture medium for several days, and then transferring the weak seedling into a strong seedling culture medium for culture, wherein the formula of the strong seedling culture medium is as follows: ms +6-BA0.5mg/L + IBA0.2mg/L; the formula of the strong seedling rooting culture medium is as follows: ms +6-BA0.1mg/L + IBA0.5mg/L. When the concentration of 6-BA in the culture medium is 0.5mg/L, IBA and the concentration is 0.2mg/L, the method is favorable for the young plant to continuously and rapidly propagate the seedling and grow to be stronger gradually; when the concentration of 6-BA0.1mg/L, IBA in the culture medium reaches 0.5mg/L, a plant can generate a large amount of fibrous roots and grow robustly.
2. Transplanting and surviving of aseptic seedlings: and (3) uncovering the sealing film at the bottleneck of the normally and robustly growing plant for indoor hardening for 2-3d, then taking out the plantlets, cleaning the culture medium attached to the plant, scattering the culture medium in a hole tray of a greenhouse, appropriately shading, spraying water in time, keeping the seedlings in a moist and air-permeable state until the plantlets adapt to a new environment and start to grow normally, counting the survival rate after 20d to 100%, and the survival rate of aseptic seedlings of other varieties can reach 100%.
Claims (7)
1. A method for tissue culture and rapid propagation of seedlings through air pineapple lateral buds is characterized by comprising the following steps:
1) collecting lateral buds of the air plants: the size of the lateral bud is preferably 1-5cm, and the cut explant is taken back to a laboratory for disinfection treatment as soon as possible;
2) Disinfection and sterilization of lateral buds and start culture: sterilizing side buds, culturing for 15-25 days in natural light to obtain a sterile side bud explant, and inoculating the treated sterile side bud explant into a start culture medium for culturing, wherein the start culture medium is MS +6-BA1-3mg/L + NAA1-2mg/L +2, 4-D0.5mg/L;
3) transferring the sterile lateral bud explant cultured in the step 2) to a cluster bud induction culture medium, culturing under 1500-2000LX light, and observing the growth and development conditions of the cluster buds, wherein the cluster bud induction culture medium is MS +6-BA1 ~ 2mg/L + NAA0.2 ~ 0.5.5 mg/L;
4) fast propagation and proliferation culture of the bud seedling, namely cutting the cluster buds obtained in the step 3) into 2-3 buds, transferring the buds into a bud seedling fast propagation and differentiation culture medium, culturing under 1500-2000LX light, observing and counting bud propagation times and bud seedling growth and development conditions after 28-30 days, wherein the bud seedling fast propagation and differentiation culture medium MS +6-BA1 ~ 2mg/L + NAA0.1 ~ 0.2.2 mg/L;
5) Strong seedling and rooting culture: transferring the cluster with 2-3 clustered seedlings into a strong seedling or strong seedling rooting culture medium, and culturing under 2000-2500LX light;
7) Hardening and transplanting aseptic seedlings.
2. The method for tissue culture and rapid propagation of seedlings through lateral buds of tillandsia undulata according to claim 1, wherein the start medium in the step 2) is MS +6-BA2mg/L + NAA2mg/L +2, 4-D0.5mg/L.
3. the method for tissue culture and rapid propagation of seedlings by using lateral buds of tillandsia undulata as claimed in claim 1, wherein the cluster bud induction medium in the step 3) is MS +6-BA1mg/L + NAA0.5mg/L.
4. The method for tissue culture and rapid propagation of seedlings through lateral buds of tillandsia as claimed in claim 1, wherein the rapid propagation and differentiation medium MS +6-BA2mg/L + NAA0.2mg/L of seedlings in the step 4).
5. The method for tissue culture and rapid propagation of seedlings by using lateral buds of tillandsia undulata as claimed in claim 1, wherein the strong seedling or rooting medium for strong seedling in step 5) is Ms +6-BA0.1 ~ 0.5mg/L + IBA0.2 ~ 0.5.5 mg/L.
6. the method for tissue culture and rapid propagation of seedlings through lateral buds of tillandsia undulata as claimed in claim 1, wherein the strong seedling culture medium of step 5) is: ms +6-BA0.5mg/L + IBA0.2mg/L.
7. The method for tissue culture and rapid propagation of seedlings through lateral buds of tillandsia undulata according to claim 1, wherein the formula of the strong seedling rooting culture medium in the step 5) is as follows: ms +6-BA0.1mg/L + IBA0.5mg/L.
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