CN111226792B - High-throughput breeding method for leaf seedlings of cymbidium sinense - Google Patents
High-throughput breeding method for leaf seedlings of cymbidium sinense Download PDFInfo
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- A—HUMAN NECESSITIES
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Abstract
The invention relates to the technical field of tissue culture and high-throughput breeding of seedlings, in particular to a high-throughput breeding method of leaf seedlings of cymbidium sinense. The method comprises the following steps: inoculating the stem base of the leaf stock plant of the variegated orchid to an MS culture medium containing 0.01-0.5 mg/L TDZ and 0.5-2.0 mg/L6-BA to induce callus, then inoculating the callus to an MS induction culture medium containing 1.0-2.5 mg/L6-BA and 0.1-0.5 mg/L NAA to induce cluster buds, and finally inoculating the cluster buds to an 1/2MS proliferation and rooting culture medium containing 1.0-2.5 mg/L6-BA and 0.1-0.5 mg/L NAA. The stem base is used as an explant material and a specific induction culture medium, so that the differentiation rate of the cluster buds is greatly improved, and the method has important significance for high-throughput breeding and genetic engineering research of future leaf seedlings of the spotted-orchid.
Description
Technical Field
The invention relates to the technical field of tissue culture and high-throughput breeding of seedlings, in particular to a high-throughput breeding method of leaf seedlings of cymbidium sinense.
Background
Leaf of variegated orchid, the academic name of Pandanus orientalis (pandanum aromaticus), the trade name of english translation (Pandan leaf), the national translation of patchouli, and also the name of variegated leaf, patchouli leaf, and slatted leaf, which are perennial evergreen horticultural crops of the genus Pandanus (pandanum) of the family of the open paphiopediatricaceae (Pandanaceae). The aristolochiaceae family has 3 kinds of plants of about 800, the plants have greatly different primary ecological environments and different economic uses, can be applied to the fields of medical treatment and cooking, belong to a plurality of species which can treat toothache and rheumatism and have wide development and utilization prospects. Wherein, the leaf of the variegated orchid is a plant with unique rice-pudding fragrance in the genus. The leaf and leaf of the variegated orchid is rich in active ingredients such as squalene, linoleic acid, estragole and sterol, has the effects of enhancing cell vitality, accelerating metabolism, improving human immunity, inhibiting cancer cell growth and the like, and is known as the vanilla of the oriental. Besides good taste, the leaf of the variegated orchid has the effects of relieving summer heat, cooling and refreshing, reducing internal heat, soothing nerves, calming, relaxing tendons and activating collaterals due to the fact that the juice of the leaf of the variegated orchid has strong antioxidant ingredients, and has very high economic development value.
The introduction of crops in China, namely the Brachypodium odoratum leaves, is originally introduced into the Xinglong area for planting, and is gradually developed to other areas in Hainan, Yunnan and other areas for planting, and the Brachypodium odoratum leaves are partially planted in provinces such as Guangdong, Taiwan and the like nowadays. The leaf juice of the southeast Asia is used as an added spice to improve the quality of cakes and beverages, and is one of common food spices for people in tropical regions. The product made of the leaf of the variegated orchid is a famous special food in tropical regions, and is popular with consumers. As a spice product which is popular among people and tropical agricultural products with important application, the problems of vigorous demand and insufficient supply exist for a long time. With the continuous improvement of the quality of life, the demand of China on the leaf of the variegated orchid is continuously increased. The leaf of the variegated orchid is taken as a typical tropical characteristic spice crop, has important significance for developing local characteristic economy and improving the income and living standard of farmers in remote areas, and has wide development prospect.
The leaf of the variegated orchid is not seen in the flower and fruit, and the traditional planting mainly comprises tillering breeding. The method has the advantages that the growth degree of the parent plant is at least more than two years, the seedlings needing tillering are limited, the requirement of the industry on high-throughput seedling breeding cannot be met, and the further development of the leaf industry of the cymbidium maculatum is restricted by the lack of the high-throughput seedling breeding technology. The tissue rapid propagation is based on the theory of cell totipotency, firstly, the maternal tissue is taken as an explant, the callus is induced under a proper environment, the maternal tissue is directly intercepted by replacing a large number of proliferation callus, then, the callus is differentiated into cluster buds, the cluster buds are divided into a plurality of bud segments, and finally, the cluster buds are rooted into a complete plant. The process can develop a plurality of individuals from one parent tissue, and greatly stimulates the reproductive capacity of the leaf tissue of the cymbidium. However, the present invention is about the rapid propagation research of leaf tissue of the cymbidium maculatum, so the technology of the present invention will fill the blank in the field.
As the leaves of the spotted-blue are not blossoming and fructification, tillering breeding is taken as the main point in the aspect of seedling breeding, the method has high requirement on the growth degree of a mother plant, the breeding efficiency of the mother plant is limited, gradual tillering breeding is needed, and the purpose of breeding seedlings in a large-scale and high-throughput manner at one time is difficult to achieve. In the aspect of genetic transformation breeding, callus composed of a plant cell collection is a good transformation vector, and regeneration of successfully transformed callus into seedlings is the key of genetic transformation breeding. Good seedling varieties often exist in a single-plant mode, and a great amount of time is needed for breeding tens of thousands of seedlings by one plant.
Disclosure of Invention
In view of the above, the invention provides a high-throughput breeding method of leaf seedlings of cymbidium sinense. The method finally realizes that only the callus is taken as the explant to carry out proliferation and differentiation, and the regenerated root is grown into a complete plant, thereby saving the primary induction time and material and simultaneously avoiding the problem of endophyte pollution in the primary induction.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a high-throughput breeding method of leaf seedlings of cymbidium sinense, which comprises the following steps:
removing leaf sheaths on the surfaces of the leaf mother plants of the cymbidium sinense, taking the base parts of stem sections close to the root ends and semi-lignified as explants, cleaning, disinfecting and sterilizing;
inoculating the sterilized explant to a callus induction culture medium for callus induction to obtain a callus; the callus induction culture medium is an MS culture medium containing 0.01-0.5 mg/L TDZ and 0.5-2.0 mg/L6-BA;
inoculating the callus onto a cluster bud induction culture medium to perform cluster bud induction to obtain cluster buds; the cluster bud induction culture medium is an MS culture medium containing 1.0-2.5 mg/L6-BA and 0.1-0.5 mg/LNAA;
after the cluster buds are divided into plants, the plants are inoculated to a proliferation and rooting culture medium for proliferation and rooting to obtain complete plants; the proliferation and rooting culture medium is 1/2MS culture medium containing 1.0-2.5 mg/L6-BA and 0.1-0.5 mg/L NAA.
Preferably, the callus induction medium is MS medium containing 0.03mg/L TDZ and 0.5 mg/L6-BA.
Preferably, the cluster bud induction medium is MS medium containing 1.0 mg/L6-BA and 0.1mg/L NAA.
Preferably, the proliferation and rooting culture medium is 1/2MS culture medium containing 1.0 mg/L6-BA and 0.1 mg/LNAA.
Preferably, callus induction conditions are 28. + -. 2 ℃ in the absence of light.
Preferably, the conditions for inducing the cluster buds are 28 +/-2 ℃, the illumination intensity is 1000-2000 lux, and the illumination period is 12 h/d.
Preferably, the light intensity induced by the cluster buds is 1500 lux.
Preferably, the conditions of proliferation and rooting are 28 +/-2 ℃, the illumination intensity is 1500-2500 lux, and the illumination period is 12 h/d.
Preferably, the light intensity for proliferating roots is 2000 lux.
Preferably, the length of the explant is 3-5 mm.
Preferably, the cleaning and sterilizing procedures of the explants are as follows: the explants are washed with detergent, then sterilized with medical alcohol and finally sterilized with mercuric chloride.
In the specific embodiment provided by the invention, the cleaning, disinfecting and sterilizing procedures of the explants are as follows: firstly, putting an explant in a container, adding 3-4 drops of a detergent, sealing with gauze, and washing for 10-30min with running water; then transferring the sample to a sterile operation table, disinfecting the sample for 30-50s by using 75% alcohol, and washing the sample for 3-4 times by using sterile water; and sterilizing for 4-8min by using 0.1% mercuric chloride, washing for 4-5 times by using sterile water, washing for 1-2min each time by using the sterile water, and draining.
The invention provides a high-throughput breeding method of leaf seedlings of cymbidium sinense. The method comprises the following steps: removing leaf sheaths on the surfaces of the leaf mother plants of the cymbidium sinense, taking the base parts of stem sections close to the root ends and semi-lignified as explants, cleaning, disinfecting and sterilizing; inoculating the sterilized explant to a callus induction culture medium for callus induction to obtain a callus; the callus induction culture medium is an MS culture medium containing 0.01-0.5 mg/L TDZ and 0.5-2.0 mg/L6-BA; inoculating the callus onto a cluster bud induction culture medium to perform cluster bud induction to obtain cluster buds; the cluster bud induction culture medium is an MS culture medium containing 1.0-2.5 mg/L of 6-BA and 0.1-0.5 mg/L of NAA; after the cluster buds are divided into plants, the plants are inoculated to a proliferation and rooting culture medium for proliferation and rooting to obtain complete plants; the proliferation and rooting culture medium is 1/2MS culture medium containing 1.0-2.5 mg/L6-BA and 0.1-0.5 mg/LNAA. The invention has the following technical effects:
the invention takes different explants and culture media as comparison methods, and the method is cultured under the same environmental conditions as the method, and the result shows that only the stem base part is taken as the explant material, the callus can be induced, the cluster buds are obtained, the tender leaves and tender stems are taken as the explant material, the callus or the cluster buds are gradually browned and died in the culture, and the callus or the cluster buds are not obtained, so the method of the invention is obviously superior to the comparison methods in the aspects of callus induction and cluster bud regeneration of the leaf callus of the cymbidium maculatum.
The method of the invention selects the side bud of the old stem as the explant, and the callus induction culture medium formula comprises the following steps: adding Thidiazuron (TDZ) and 6-benzylaminopurine (6-BA) by taking MS as a basic culture medium; the formula of the cluster bud induction culture medium comprises: adding 6-benzylamino adenine (6-BA) and Naphthalene Acetic Acid (NAA) by taking MS as a basic culture medium; the formula of the culture medium for proliferating and rooting the cluster buds is as follows: 1/2MS is used as a basic culture medium, and 6-benzylaminopurine (6-BA) and naphthylacetic acid (NAA) are added, so that the differentiation rate of more than 18-25 cluster buds can be achieved by culturing the base of one stem segment, and the method has important significance for high-throughput breeding and genetic engineering research of future leaf seedlings of the melaleuca sinica.
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FIG. 1 is the tissue culture and rapid propagation method of leaf seedlings of the spotted-blue.
Detailed Description
The invention discloses a high-throughput breeding method of leaf seedlings of cymbidium sinense, and a person skilled in the art can appropriately improve process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
The invention relates to the technical field of tissue culture and high-throughput breeding of spotted-blue leaves, and discloses a seedling rapid propagation technical method taking the base parts of lateral buds of spotted-blue leaves as explants. The method of the invention comprises the steps of picking off buds of about 0.5-0.8cm grown from the old stems of the leaves of the cymbidium sinense, and culturing for about 30 days after disinfection and sterilization to induce callus; the callus is first proliferated to 0.5cm in diameter to induce the callus to differentiate into multiple shoots, and the multiple shoots are further proliferated and elongated to grow root into complete plant for high throughput breeding. Firstly, in 25-35 days of culture on the MS culture medium containing TDZ and 6-BA, the periphery of the base part of the lateral bud can be expanded to generate yellow callus, and then the callus is further proliferated to be yellow green on the MS culture medium containing 6-BA and NAA, and is piled into a mass and a hard callus block. Wherein the callus blocks are subcultured on MS culture medium containing 6-BA and NAA for 30-40 days, the callus blocks begin to brown slightly and then a large amount of cluster buds begin to be extracted, the cluster buds are inoculated into 1/2MS culture medium containing only NAA for rooting culture to complete plants, and the plants can be transplanted into soil. The method finally realizes that only the callus is taken as the explant to carry out proliferation and differentiation, and the regenerated root is grown into a complete plant, thereby saving the primary induction time and material and simultaneously avoiding the problem of endophyte pollution in the primary induction. The in vitro regeneration method of the spotted-orchid leaves provides technical support for the industrial high-throughput breeding and genetic engineering breeding research of the spotted-orchid leaf seedlings in the future.
The culture medium formula used in the high-throughput breeding method of the leaf seedlings of the cymbidium sinense provided by the invention can be purchased from the market.
The invention is further illustrated by the following examples:
example 1: the method of the invention
Cutting a parent plant, poking leaf sheaths on the upper surface of the leaf parent plant of the cymbidium sinense layer by layer until the semi-lignified stem base part close to the root end is exposed, and then cutting the semi-lignified stem segment base part with the length of 3-5 mm. Firstly, the explant is put into a triangular flask, 3-4 drops of detergent are added, and the explant is sealed by clean gauze and washed by running water for 10-30min until the surface of the explant is washed clean by foams. And then transferring to a sterile operation table, sterilizing the surface of the sterile operation table by using 75% alcohol for 30-50s, then washing the sterile operation table for 3-4 times, then sterilizing the sterile operation table for 4-8min by using 0.1% mercuric chloride, washing the sterile operation table for 4-5 times by using sterile water for 1-2min each time, and draining the water for later inoculation.
Placing the sterilized stem base on a callus induction culture medium, and culturing in the dark at 28 +/-2 ℃ to induce the callus, wherein the culture medium formula is MS +0.03mg/L Thidiazuron (TDZ) +0.5 mg/L6-benzylamino adenine (6-BA).
And inoculating the induced callus to a cluster bud induction culture medium in small blocks for cluster bud induction, and culturing at the temperature of 28 +/-2 ℃ under 1500lux illumination, wherein the formula of the culture medium is MS +1.0 mg/L6-benzylamino adenine (6-BA) +0.1mg/L naphthylacetic acid (NAA).
The induced cluster buds are divided into plants and inoculated to a proliferation and rooting culture medium, and proliferation and rooting are carried out by 2000lux illumination culture at the temperature of 28 +/-2 ℃, and the formula of the culture medium is 1/2MS +1.0 mg/L6-benzylamino adenine (6-BA) +0.1mg/L naphthylacetic acid (NAA).
The culture effect of each period in the tissue culture rapid propagation technology of the embodiment is shown in figure 1.
Comparative example 1:
the tender leaves and tender stems of the leaf of the cymbidium sinensis are taken as explants, and the culture method in the embodiment 1 is adopted, and the method comprises the following concrete steps:
taking tender leaves and tender stems of the leaf stock plant of the cymbidium goeringii as explant materials. Firstly, the explant is put into a triangular flask, 3-4 drops of detergent are added, and the explant is sealed by clean gauze and washed by running water for 10-30min until the surface of the explant is washed clean by foams. And then transferring to an aseptic operation table, sterilizing the surface of the aseptic operation table by using 75% alcohol for 30-50s, washing the aseptic operation table with sterile water for 3-4 times, sterilizing the aseptic operation table with 0.1% mercuric chloride for 4-8min, washing the aseptic operation table with sterile water for 4-5 times, washing the aseptic operation table with sterile water for 1-2min each time, draining water and then waiting for inoculation.
Placing sterilized young leaves on a callus induction culture medium, and culturing in the dark at 28 +/-2 ℃ to induce the callus, wherein the formula of the culture medium is MS +0.03mg/L Thidiazuron (TDZ) +0.5 mg/L6-benzylamino adenine (6-BA).
And inoculating the induced callus on a cluster bud induction culture medium in small blocks for cluster bud induction, and culturing at the temperature of 28 +/-2 ℃ under the illumination intensity of 1500lux, wherein the formula of the culture medium is MS +1.0 mg/L6-benzylamino adenine (6-BA) +0.1mg/L naphthylacetic acid (NAA).
The induced cluster buds are divided into plants and inoculated to a proliferation and rooting culture medium, and proliferation and rooting are carried out by 2000lux illumination culture at the temperature of 28 +/-2 ℃, and the formula of the culture medium is 1/2MS +1.0 mg/L6-benzylamino adenine (6-BA) +0.1mg/L naphthylacetic acid (NAA).
Comparative example 2:
the callus induction medium in example 1 was changed to MS +0.1 mg/L2, 4-dichlorophenoxyacetic acid (2,4-D) +0.5mg/L Kinetin (KT), and the clumpy shoot induction medium was changed to MS +1.0mg/L Kinetin (KT) +0.1mg/L naphthylacetic acid (NAA). The method comprises the following specific steps:
cutting a parent plant, poking leaf sheaths on the upper surface of the leaf parent plant of the cymbidium sinense layer by layer until the semi-lignified stem base part close to the root end is exposed, and then cutting the semi-lignified stem segment base part with the length of 3-5 mm. Firstly, the explant is put into a triangular flask, 3-4 drops of detergent are added, and the explant is sealed by clean gauze and washed by running water for 10-30min until the surface of the explant is washed clean by foams. And then transferring to an aseptic operation table, sterilizing the surface of the aseptic operation table by using 75% alcohol for 30-50s, washing the aseptic operation table with sterile water for 3-4 times, sterilizing the aseptic operation table with 0.1% mercuric chloride for 4-8min, washing the aseptic operation table with sterile water for 4-5 times, washing the aseptic operation table with sterile water for 1-2min each time, draining water and then waiting for inoculation.
Placing the sterilized stem base on a callus induction culture medium, and culturing in the dark at 28 + -2 deg.C for callus induction, wherein the culture medium formula is MS +0.1 mg/L2, 4-dichlorophenoxyacetic acid (2,4-D) +0.5mg/L Kinetin (KT).
Inoculating the induced callus to a cluster bud induction culture medium in small pieces for cluster bud induction, and culturing at the temperature of 28 +/-2 ℃ and the illumination intensity of 1500lux, wherein the formula of the culture medium is MS +1.0mg/L Kinetin (KT) +0.1mg/L naphthylacetic acid (NAA).
The induced cluster buds are divided into plants and inoculated to a proliferation and rooting culture medium, and proliferation and rooting are carried out by 2000lux illumination culture at the temperature of 28 +/-2 ℃, and the formula of the culture medium is 1/2MS +1.0 mg/L6-benzylamino adenine (6-BA) +0.1mg/L naphthylacetic acid (NAA).
Comparative example 3:
the callus induction medium in example 1 was changed to MS +0.1 mg/L2, 4-dichlorophenoxyacetic acid (2,4-D) +0.5mg/L Kinetin (KT), and the rooting medium was changed to 1/2MS +1.0mg/L Kinetin (KT) +0.1mg/L naphthylacetic acid (NAA). The method comprises the following specific steps:
taking tender leaves and tender stems of the leaf stock plant of the cymbidium goeringii as explant materials. Firstly, the explant is put into a triangular flask, 3-4 drops of detergent are added, and the explant is sealed by clean gauze and washed by running water for 10-30min until the surface of the explant is washed clean by foams. And then transferring to an aseptic operation table, sterilizing the surface of the aseptic operation table by using 75% alcohol for 30-50s, washing the aseptic operation table with sterile water for 3-4 times, sterilizing the aseptic operation table with 0.1% mercuric chloride for 4-8min, washing the aseptic operation table with sterile water for 4-5 times, washing the aseptic operation table with sterile water for 1-2min each time, draining water and then waiting for inoculation.
Placing sterilized young leaves on a callus induction culture medium, and culturing in the dark at 28 +/-2 ℃ to induce the callus, wherein the culture medium formula is MS +0.1 mg/L2, 4-dichlorophenoxyacetic acid (2,4-D) +0.5mg/L Kinetin (KT).
And inoculating the induced callus to a cluster bud induction culture medium in small blocks for cluster bud induction, and culturing at the temperature of 28 +/-2 ℃ under 1500lux illumination, wherein the formula of the culture medium is MS +1.0 mg/L6-benzylamino adenine (6-BA) +0.1mg/L naphthylacetic acid (NAA).
The induced cluster buds are subjected to plant division, inoculated to a proliferation and rooting culture medium, and cultured by illumination intensity of 2000lux at the temperature of 28 +/-2 ℃ for proliferation and rooting, wherein the formula of the culture medium is 1/2MS +1.0mg/L Kinetin (KT) +0.1mg/L naphthylacetic acid (NAA).
Comparative example 4:
the callus induction medium in example 1 was changed to MS +0.1 mg/L2, 4-dichlorophenoxyacetic acid (2,4-D) +0.5 mg/L6-BA. The method comprises the following specific steps:
cutting a parent plant, poking leaf sheaths on the upper surface of the leaf parent plant of the cymbidium sinense layer by layer until the semi-lignified stem base part close to the root end is exposed, and then cutting the semi-lignified stem segment base part with the length of 3-5 mm. Firstly, the explant is put into a triangular flask, 3-4 drops of detergent are added, and the explant is sealed by clean gauze and washed by running water for 10-30min until the surface of the explant is washed clean by foams. And then transferring to an aseptic operation table, sterilizing the surface of the aseptic operation table by using 75% alcohol for 30-50s, washing the aseptic operation table with sterile water for 3-4 times, sterilizing the aseptic operation table with 0.1% mercuric chloride for 4-8min, washing the aseptic operation table with sterile water for 4-5 times, washing the aseptic operation table with sterile water for 1-2min each time, draining water and then waiting for inoculation.
Placing the sterilized stem base on callus induction culture medium, and performing light-shielding culture at 28 + -2 deg.C for callus induction, wherein the culture medium formula is MS +0.1 mg/L2, 4-dichlorophenoxyacetic acid (2,4-D) +0.5 mg/L6-BA.
And inoculating the induced callus on a cluster bud induction culture medium in small blocks for cluster bud induction, and culturing at the temperature of 28 +/-2 ℃ under the illumination intensity of 1500lux, wherein the formula of the culture medium is MS +1.0 mg/L6-benzylamino adenine (6-BA) +0.1mg/L naphthylacetic acid (NAA).
The induced cluster buds are divided into plants and inoculated to a proliferation and rooting culture medium, and proliferation and rooting are carried out by 2000lux illumination culture at the temperature of 28 +/-2 ℃, and the formula of the culture medium is 1/2MS +1.0 mg/L6-benzylamino adenine (6-BA) +0.1mg/L naphthylacetic acid (NAA).
Test example 1: comparative test
The test was conducted in accordance with the methods of example 1 and comparative examples 1 to 4, and the callus induction rate and the cluster bud induction rate were counted, and the results are shown in Table 1.
TABLE 1 comparison of callus induction rate and cluster bud induction rate for different culture methods
As can be seen from Table 1, the proliferation rate of the method for obtaining the complete plant by regenerating the explant from the lateral bud base on the old stem is more than 100 times. By adopting the culture method of callus induction and cluster bud differentiation provided by the invention, the callus induction rate reaches 80%, and the cluster bud differentiation rate reaches about 92%, so that the culture method is an optimal culture method compared with other control methods.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. A high-throughput breeding method of leaf seedlings of cymbidium sinense is characterized by comprising the following steps:
removing leaf sheaths on the surfaces of the leaf parent plants of the cymbidium sinense, taking the base parts of the lateral buds of the stem segments close to the root ends and semi-lignified as explants, and cleaning, disinfecting and sterilizing the explants;
inoculating the sterilized explant to a callus induction culture medium for callus induction to obtain a callus; the callus induction culture medium is an MS culture medium containing 0.01-0.5 mg/L TDZ and 0.5-2.0 mg/L6-BA;
inoculating the callus onto a cluster bud induction culture medium to perform cluster bud induction to obtain cluster buds; the cluster bud induction culture medium is an MS culture medium containing 1.0-2.5 mg/L6-BA and 0.1-0.5 mg/LNAA;
after the cluster buds are divided into plants, the plants are inoculated to a proliferation and rooting culture medium for proliferation and rooting to obtain complete plants; the proliferation and rooting culture medium is 1/2MS culture medium containing 1.0-2.5 mg/L6-BA and 0.1-0.5 mg/L NAA.
2. The high-throughput breeding method of claim 1, wherein the callus induction medium is MS medium containing 0.03mg/L TDZ and 0.5 mg/L6-BA.
3. The high-throughput breeding method of claim 1, wherein the multiple shoot induction medium is MS medium containing 1.0 mg/L6-BA and 0.1mg/L NAA.
4. The high-throughput breeding method of claim 1, wherein the rooting-for-proliferation medium is 1/2MS medium containing 1.0 mg/L6-BA and 0.1 mg/LNAA.
5. The high-throughput breeding method according to claim 1, wherein the callus induction conditions are 28 ± 2 ℃ and are protected from light.
6. The high-throughput breeding method according to claim 1, wherein the conditions for inducing the multiple shoots are 28 ± 2 ℃, the illumination intensity is 1000-2000 lux, and the illumination period is 12 h/d.
7. The high-throughput breeding method according to claim 1, wherein the conditions for proliferation and rooting are 28 +/-2 ℃, the illumination intensity is 1500-2500 lux, and the illumination period is 12 h/d.
8. The high-throughput breeding method of claim 1, wherein the length of the explant is 3-5 mm.
9. The high-throughput breeding method according to claim 1, wherein the cleaning and sterilizing procedures of the explants are as follows: the explants are washed with detergent, then sterilized with medical alcohol and finally sterilized with mercuric chloride.
10. The high-throughput breeding method of claim 9, wherein the cleaning and sterilizing process of the explants is as follows: firstly, putting an explant in a container, adding 3-4 drops of a detergent, sealing with gauze, and washing for 10-30min with running water; then transferring the sample to a sterile operation table, disinfecting the sample for 30-50s by using 75% alcohol, and washing the sample for 3-4 times by using sterile water; and sterilizing for 4-8min by using 0.1% mercuric chloride, washing for 4-5 times by using sterile water, washing for 1-2min each time by using the sterile water, and draining.
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| CN112931219B (en) * | 2021-04-12 | 2022-05-31 | 海南热作高科技研究院有限公司 | Tissue culture and rapid propagation method of Pandanus communis |
| CN113179951B (en) * | 2021-06-02 | 2022-04-29 | 中国热带农业科学院橡胶研究所 | Rapid breeding method for tissue culture seedlings of Pandanum amazonicum |
| CN115119747A (en) * | 2022-04-30 | 2022-09-30 | 中国热带农业科学院海口实验站 | Leaf tissue culture medium of cymbidium maculatum and tissue culture rapid propagation method |
| CN114847161B (en) * | 2022-05-12 | 2023-05-02 | 中国热带农业科学院香料饮料研究所 | Method for improving transplanting survival rate and fragrance of cymbidium She Zupei seedlings |
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