CN110547196A - Method for quickly propagating air pineapple seedlings through leaf tissue culture - Google Patents
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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 quickly propagating air pineapple seedlings by leaf group culture, which comprises the following steps: 1) taking an air pineapple leaf explant; 2) sterilizing the leaf explants; 3) inducing the cluster buds of the leaf explants; 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 wide material source, quick and convenient material taking, simple operation, high propagation coefficient, high seedling growth speed, capability of quickly propagating a large number of tissue culture seedlings with consistent properties, consistent growth and robustness in a short time and the like, solves the defects that the air pineapple seeds are difficult to propagate, the offspring is possible to have variation and can not keep the excellent properties of the female parent, and also solves the practical problems that a plurality of rare seeds or varieties can generate rare sub-plants only after flowers are bloomed and can not meet the requirement of tissue culture and quick propagation for collecting explant materials all the year round.
Description
Technical Field
The invention relates to the field of plant breeding, in particular to a method for quickly breeding air pineapple seedlings by leaf group culture.
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.
The main factors restricting the development and popularization of the air pineapple are fewer seedlings and higher price, especially some rare provenances. The main three propagation modes of the air pineapple are as follows: plant division propagation, seed propagation and tissue culture (Dingkui et al, 2010). The method is the most common method, generally, one to a plurality of lateral buds can grow from a robust mother plant after flowers, the separation is optimal when the lateral buds grow to 1/3-1/2 of the size of the mother plant, but the number of the sub-plants is easily influenced by factors such as varieties, cultivation environment and the like, so the number of the sub-plants of the plant division propagation is limited, and the propagation coefficient is very low; the sowing and propagation firstly need to wait for the seed pod to mature, the seed pod maturation generally needs 3-30 months, and different varieties have great difference; in addition, whether the seeds can germinate and whether the seedlings can normally grow can also be easily influenced by the environment such as temperature, humidity, ventilation and the like, the seedlings are very fragile in the initial stage, the growth is very slow, and the seedlings are very easy to die; therefore, the difficulty of seed propagation and production is high; in the seed propagation, the characters of hybrid progeny are easy to separate, the excellent characters of the female parent are difficult to maintain, and the uniformity and consistency of progeny groups are directly influenced. Plant tissue culture provides an effective way for the propagation of the air pineapples, and at present, research reports on the aspect of tissue culture and rapid propagation of the air pineapples are very few, and particularly, the tissue culture and rapid propagation by utilizing the leaves (with leafstalks) of the air pineapples 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 Lipeng discloses a tissue culture and rapid propagation method of tillandsia, which is mainly characterized in that 4-5 seedlings can be separated from the germination of one seed to the final separation of the sterile seed. It can be seen that the above reports are different from the present invention in terms of explant, culture medium formulation ratio, method and technology, and the experimental results and effects are also different (or completely). Extremely low propagation coefficient and extremely slow growth habit of seedlings, which greatly increases the difficulty of commercial production and industrial development of the air pineapple, particularly the excellent rare germplasm resources.
Disclosure of Invention
the purpose of the invention is as follows: the invention provides a method for quickly propagating air pineapple seedlings by tissue culture of leaves (with petioles) 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 quickly propagating air pineapple seedlings by leaf group culture, which comprises the following steps:
1) The air pineapple leaf explants are adopted: the complete young leaves which grow well and have no plant diseases and insect pests of the mother plant are required to be adopted; when the leaves are collected, the leaves are prevented from being kept in plum rain season as much as possible or kept sunny for at least 3 days, and the 3 rd to 6 th young leaves with best leaf stalks of the parent plant are selected as the leaf explants;
2) sterilizing and starting culture of the leaf explants: soaking the leaf explants in washing powder for 5-8min, then washing in running water for 30-50min, then sterilizing with a mixed disinfectant prepared from 1% sodium hypochlorite and 2-3 drops of Tween for 5-8min, washing with sterilized water for 3 times, then sucking off surface water, taking the leaves to a super clean workbench, soaking the leaves in 70-75% alcohol for 30s, then placing the leaves in 0.1% mercuric chloride for sterilizing for 8-10min, gently shaking the bottles during the soaking, washing with sterilized water for 8-10 times after sterilizing, wiping off the water with sterilized absorbent paper, then cutting the leaves into 1-2cm strips with leaf stalks, spreading and inoculating the strips on a start-up culture medium, carrying out dark culture for about 15-25d to count the contamination rate of the leaf explants and observing the growth and development conditions of leaf callus; the bacterial contamination rate is the seed number of the bacterial contamination/the seed number of the test, 100 percent;
3) induced differentiation of leaf explants clumpy buds: transferring the sterile leaf explants obtained in the step 2) from a starting culture medium to a cluster bud induced differentiation culture medium, culturing for 28-56 days under visible light, and observing the growth and development conditions of the cluster buds;
4) And (3) rapid propagation and multiplication culture of the bud seedlings: transferring the cluster buds obtained in the step 3) to a bud seedling rapid propagation and proliferation culture medium, culturing for about 28 days under 1500-2000LX light, counting the multiplication times of leaf buds and observing the growth and development conditions of the bud seedlings;
5) Strong seedling and rooting culture: transferring the bud seedling into a strong seedling rooting culture medium after 28-30 days to grow into a normal strong plant with a plurality of stems and leaves;
6) hardening and transplanting aseptic seedlings: and (3) uncovering the sealing film of the bottleneck of the plant growing normally and robustly in the step 5) for indoor hardening for 2-3d, then taking out the plantlets, cleaning the culture medium attached to the plants, scattering the plantlets in a cool and ventilated hole tray of a greenhouse, appropriately shading, spraying a small amount of water in time, keeping the seedlings in a relatively humid environment with good air permeability until the plantlets adapt to a new environment and start to grow normally, and counting the survival rate to 100% after 20 d.
Wherein the starting culture medium in the step 2) is MS +6-BA0.01-0.1mg/L + NAA1-2mg/L +2, 4-D1-4 mg/L.
Wherein the cluster bud induction culture medium in the step 3) is MS +6BA0.5mg/L + KT0.5mg/L + TDZ0.5 mg/L + NAA0.5mg/L + CH300 mg/L.
Wherein the bud seedling rapid propagation and differentiation culture medium in the step 4) is MS +6BA2mg/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.
All the above culture temperatures are between 20-28 ℃.
According to the invention, the formula of the starting culture medium with the best leaves is obtained by research and analysis, wherein the formula is MS +6-BA0.01+ NAA1.0+2, 4-D3.0, and the formula is preferably the medium callus degree of the leaves; the best culture medium for inducing differentiation of the fascicular bud is MS +6BA0.5mg/L + KT0.5mg/L + TDZ0.5 mg/L + NAA0.5mg/L + CH300mg/L for culturing 56d, and the fascicular bud has the highest occurrence degree; the best formula of the culture medium for rapid propagation and proliferation of the leaf buds is MS +6-BA2.0mg/L + NAA0.2mg/L, and the multiplication times of the buds are about 10 times. The leaf can be propagated to about 10 buds on average about 120 days after the leaf (with the petiole) is cultured by the rapid propagation starting method, the propagation coefficient is obviously higher than that of the conventional propagation method, the seedling growth speed is high, the seedling is robust, and a large number of tissue culture seedlings with consistent properties, growth and robustness can be rapidly propagated in a short time.
Has the advantages that: compared with the prior art, the invention has the following advantages: the method has the advantages of wide material source, rapid and convenient material taking, simple operation, high propagation coefficient, fast seedling growth speed, capability of rapidly propagating a large number of tissue culture seedlings with consistent properties, consistent growth and robustness in a short time, and the like. The technology not only solves the defects that the seeds of the tillandsia are difficult to propagate, the offspring is possible to generate variation and the excellent characters of the female parent can not be maintained, but also solves the practical problems that a plurality of rare seeds or varieties can generate rare sub-plants (lateral buds) only after flowers are blossomed and can not meet the requirement of tissue culture and rapid propagation for collecting explant materials all the year round. Therefore, the invention is not only beneficial to large-scale industrialized production of excellent rare germplasm, but also provides an effective way for commercial production and industrial development of the tillandsia.
Drawings
FIG. 1 schematic representation of leaf-based callus induction;
FIG. 2 is a schematic diagram of induced differentiation culture of leaf-based multiple shoots;
FIG. 3 is a schematic diagram of propagation and rapid propagation of sprouts;
FIG. 4 is a schematic diagram of rooting culture of strong seedlings.
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.
Example 1: selection, disinfection and sterilization of air pineapple leaves
1. The air pineapple leaf explants are adopted: the material taking time is preferably selected in the morning of breeze in sunny days and cool weather; the preferred varieties are: tricolor (ludwigia prostrata) t.tricolor, beji t.bergeri, harris t.harrisii, trichrome t.juncea, genine t.lontha, etc. The selected plants need to grow strongly without any plant diseases and insect pests, in order to ensure high survival rate, the knife for cutting the leaves needs high-temperature disinfection and sterilization in advance, the knife edge needs to be sharp, 3-6 young leaves with leaf stalks of the parent plants are cut, and the young leaves are quickly brought back to a laboratory for disinfection treatment.
2. Sterilizing the leaf explants: soaking the T.juncea leaf explant in washing powder for 5-8min, and washing in running water for 30-50 min. Then 1% sodium hypochlorite and 2-3 drops of Tween are prepared into a mixed disinfectant to be disinfected for 5-8min, the disinfectant is washed by sterilized water for 3 times, then surface moisture is absorbed, the disinfectant is taken on a super-clean workbench, the disinfectant is firstly soaked by 70-75% alcohol for 30s, then the disinfectant is put into 0.1% mercuric chloride to be disinfected for 8-10min, the bottle can be gently shaken in the period, the disinfectant and sterilized aseptic water is washed by sterilized water for 8-10 times, the water is wiped by sterilized absorbent paper, and then the leaves are cut into 1-2cm strips with petioles and are tiled and inoculated on different start-up culture media; in order to reduce cross infection, 1-2 leaf explants are placed in each bottle, 20 bottles are inoculated together, dark culture is carried out for 15-25d, and the bacterial contamination rate of the leaf explants and the growth and development conditions of leaf buds are counted, and the results are shown in Table 1.
TABLE 1 Effect of different start-up medium formulations on the leaf contamination rate and the degree of leaf callus formation
as can be seen from fig. 1 and table 1: the infection rate of all the formula leaves is very low, and the influence on the growth and development of the leaf sprouts is very small, which indicates that the designed disinfection method is suitable and effective for sterilizing the leaf explants, and the loss of leaf cell contents and the damage to leaf tissues can not be caused; therefore, the main influences on the growth and development of leaf buds are the hormone ratios in different starting culture medium formulas; the reaction of the leaves to different starting culture medium formulas is whether callus is formed at the leaf base petioles and the growth and development states of the callus are formed; the analysis shows that except the Y01 formula, the leaves do not react, other formulas can form callus at the basal petioles of the leaves, but the callus degree is greatly different, wherein the formulas are as high as Y2, Y3, Y4, Y6, Y7 and Y8, but the explant is more beneficial to the induction of the buds of the leaf cluster and the multiplication of the leaf buds, and the explant needs to be transferred to a rapid propagation medium for the induction and the differentiation of the buds of the lower leaf cluster and the continuous culture and observation of the buds.
example 2 induced differentiation of shoot of leaf plexus and Rapid propagation and proliferation culture of shoot
1. Induced differentiation culture of leaf cluster buds: the sterile leaf explants obtained in example 1 were transferred to a leaf cluster bud differentiation induction medium, cultured for about 28d or 56d at 14h/d under visible light, to induce the generation of leaf cluster buds, and the growth and development of the cluster buds were observed, and the results are shown in FIG. 2 and Table 2.
TABLE 2 influence of the formula of the culture medium for inducing differentiation of the cluster buds on the growth and development of the leaf cluster buds
as can be seen from table 2: except that Y01 is a control, no cluster buds occur under the influence of the formula of the starting culture medium, all other serial numbers can induce cluster buds on the cluster bud induction culture medium, but the cluster bud occurrence degree is greatly different; further research and analysis show that the difference of the cluster buds is mainly influenced by the formula of the starting culture medium and also related to the days of culture transferred to the cluster bud induction culture medium, wherein the culture of 56D is better than the culture of 28D generally, the cluster buds have higher generation degree, the highest generation degree of the cluster buds is the Y3 serial number of the culture of 56D, namely Y3 cultures 56D on the culture medium of MS +6-BA0.5+ KT0.5+ NAA0.5, and the cluster buds have the highest generation degree, so that the best starting culture medium is the formula of MS +6-BA0.01+ NAA1.0+2, 4-D3.0, and the leaf callus degree is medium; the best culture medium for inducing differentiation of the leaf cluster buds is MS +6BA0.5+ KT0.5+ TDZ0.5+ NAA0.5+ CH300 formula culture 56d, and the cluster buds have the highest occurrence degree.
2. and (3) proliferation and rapid propagation culture of the bud seedlings: the sterile leaf cluster buds obtained in the example 2 are transferred to a leaf bud seedling multiplication rapid culture medium, namely MS +6BA2.0mg/L + NAA0.2mg/upper, and cultured under 1500-2000LX light for about 14h/d, and statistical bud multiplication times are observed after 28-30 days, and the results are shown in a graph 3 and a table 3.
TABLE 3 influence of the culture formula for the Rapid propagation of sprouts on sprout proliferation
as seen from table 3: the multiplication times of buds are greatly influenced by the results in the table 2, except that Y01 has no multiplication of buds as a control, materials with other serial numbers grow well when being transferred to a bud seedling rapid propagation culture medium, but the multiplication times of buds are greatly different, the best serial number is Y3 obtained by statistics, and the multiplication times of buds on a bud rapid propagation culture medium formula MS +6-BA2.0+ NAA0.2 can reach 10 times; further research and analysis show that the formula of the starting culture medium with the best leaves is MS +6-BA0.01+ NAA1.0+2, 4-D3.0, and the leaf callus degree is medium; the best culture medium for inducing differentiation of the leaf cluster buds is MS +6BA0.5+ KT0.5+ TDZ0.5+ NAA0.5+ CH300 formula culture 56d, and the cluster buds have the highest occurrence degree; the best formula of the culture medium for rapid propagation and proliferation of the leaf buds is MS +6-BA2.0+ NAA0.2, and the multiplication times of the buds are about 10 times. The leaf can be propagated to about 10 buds about 120 days on average by one leaf with petiole through the rapid propagation starting culture, the propagation coefficient is obviously higher than that of the conventional propagation method, the seedling growth speed is high, the seedling is robust, and a large number of tissue culture seedlings with consistent properties, consistent growth and robustness can be rapidly propagated in a short time.
EXAMPLE 3 Strong seedling rooting culture and transplantation of the seedlings
1. strong seedling and rooting culture: the fast-propagating bud seedlings obtained in the example 2 are divided into 2-3 clusters and transferred into a strong seedling or strong seedling rooting culture medium, and the clusters are cultured for 14h/d under the light of 2000-2500LX, and the small bud seedlings can grow into normal and robust plants with a plurality of roots, stems and leaves in 100% in about 28 days (figure 4). Directly transferring the plant with better growth condition into a strong seedling culture medium for culture according to the condition of the bud seedling, firstly culturing the plant with weaker growth vigor in a strong seedling rooting culture medium for several days, then transferring the plant into a strong seedling culture medium for culture, wherein the formulas of the strong seedling culture medium or the strong seedling rooting culture medium are respectively as follows: ms +6-BA0.5mg/L + IBA0.2mg/L; or: ms +6-BA0.1mg/L + IBA0.5mg/L.
2. Transplanting and surviving of aseptic seedlings: and (3) uncovering the sealing film of the bottleneck of the plant growing normally and robustly for indoor hardening for 2-3d, then taking out the plantlets, cleaning the culture medium attached to the plants, 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, and counting the survival rate to 100% after 20 d. The experimental results of other varieties of the invention are similar to the results of the big tricolor T.juncea, and the survival rate of the invention can also reach 100%.
3. All the culture temperatures are 20-28 ℃, and the culture medium pH is 5.8, the sucrose content is 30g/L, and the agar content is 6.5g/L, which is not specifically shown.
Claims (6)
1. A method for quickly propagating tillandsia seedlings through leaf group culture is characterized by comprising the following steps:
1) The air pineapple leaf explants are adopted: the complete young leaves which grow well and have no plant diseases and insect pests of the mother plant are required to be adopted; when the leaves are collected, the leaves are prevented from being kept in plum rain season as much as possible or kept sunny for at least 3 days, and the 3 rd to 6 th young leaves with best leaf stalks of the parent plant are selected as the leaf explants;
2) Sterilizing and starting culture of leaf explants, namely soaking the leaf explants in washing powder for 5-8min, then washing the leaf explants in inflow water for 30-50min, then sterilizing the leaf explants by using 1% of sodium hypochlorite and 2-3 drops of Tween to prepare a mixed disinfectant for 5-8min, washing the sterilized water for 3 times, then sucking surface water, taking the leaf explants onto a super-clean workbench, soaking the leaf explants in 70-75% of alcohol for 30s, then putting the leaf explants in 0.1% of mercury bichloride for sterilizing for 8-10min, slightly shaking the bottles during the sterilization, washing the sterilized water for 8-10 times after sterilization, wiping the water by using sterilized absorbent paper, then cutting the leaves into 1-2cm long strips with leaf stalks, spreading the long strips and inoculating the long strips on a starting culture medium, and carrying out dark culture for 15-25D to obtain sterile leaves or leaf callus, wherein the starting culture medium is MS +6-BA0.01-0.1mg/L + NAA1-2mg/L +2,4-D1 ~ 4 mg/L;
3) Induced differentiation of leaf explants clumpy buds: transferring the sterile leaf or leaf callus of the step 2) from a starting culture medium to a cluster bud induction culture medium, culturing under visible light, and observing the growth and development conditions of the cluster buds;
4) And (3) rapid propagation and multiplication culture of the bud seedlings: transferring the cluster buds obtained in the step 3) into a bud seedling rapid propagation and differentiation culture medium, and culturing under 1500-2000LX light;
5) Strong seedling and rooting culture: transferring the bud seedling into a strong seedling rooting culture medium after 28-30 days to grow into a normal strong plant with a plurality of stems and leaves;
6) Hardening and transplanting aseptic seedlings.
2. The method for rapid propagation of tillandsia through leaf mass culture as recited in claim 1, wherein the start-up medium in step 2) is MS +6-BA0.01mg/L + NAA1mg/L +2,4-D3 mg/L.
3. The method for rapid propagation of air pineapple seedlings through leaf group culture as claimed in claim 1, wherein the cluster bud induction medium in the step 3) is MS +6BA0.5mg/L + KT0.5mg/L + TDZ0.5 mg/L + NAA0.5mg/L + CH300 mg/L.
4. The method for rapid propagation of air plant pear seedlings through leaf group culture according to claim 1, wherein the rapid propagation and differentiation medium of the sprout in the step 4) is MS +6BA2mg/L + NAA0.2mg/L.
5. The method for rapid propagation of tillandsia seedlings by leaf mass culture as claimed in claim 1, wherein the rooting medium for strong seedlings in step 5) is Ms +6-BA0.1 ~ 0.5mg/L + IBA0.2 ~ 0.5.5 mg/L.
6. The method for rapid propagation of air plant pear seedlings by leaf group culture as claimed in claim 1, wherein the step 6) hardening transplantation of aseptic seedlings comprises: and (3) uncovering the sealing film of the bottle mouth of the plant growing normally and robustly in the step 5) for indoor seedling hardening for 2-3d, then taking out the plantlets, cleaning the culture medium attached to the plants, scattering the plantlets in a cool and ventilated hole tray of a greenhouse, appropriately shading, spraying a small amount of water in time, keeping the seedlings in a relatively humid environment with good air permeability until the plantlets adapt to a new environment and start to grow normally.
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| CN113575422A (en) * | 2021-09-02 | 2021-11-02 | 中国科学院分子植物科学卓越创新中心 | Efficient in-vitro regeneration method of pineapple leaves |
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