CN110604054B - High-throughput breeding method for seedlings of citronella - Google Patents
High-throughput breeding method for seedlings of citronella Download PDFInfo
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Abstract
The invention relates to the technical field of high-throughput breeding of seedlings, in particular to a high-throughput breeding method of a seedling of a citronella. The method comprises the following steps: removing leaf sheath on the surface of the parent plant of the citronella, taking the base of the stem section which is close to the root end and semi-lignified as an explant, cleaning, disinfecting and sterilizing; inoculating the sterilized explant to a callus induction culture medium for callus induction to obtain a callus; inoculating the callus onto a cluster bud induction culture medium to perform cluster bud induction to obtain cluster buds; and (4) after the cluster buds are divided into plants, inoculating the plants to a proliferation and rooting culture medium for proliferation and rooting to obtain complete plants. The invention realizes that the base of one stem segment can have the differentiation rate of more than 18-25 cluster buds through culture, and has important significance for high-throughput breeding and genetic engineering research of future citronella seedlings.
Description
Technical Field
The invention relates to the technical field of high-throughput breeding of seedlings, in particular to a high-throughput breeding method of a seedling of a citronella.
Background
Citronella (Cymbopogon winneriana Jowitt), alias: java citronella, which belongs to Gramineae and citronella and belongs to perennial large-scale symposium herbaceous plants, has strong fragrance; the root system is shallow, the rhizome is thick and short, the texture is hard, and the tillering capacity is strong. The leaf sheath is wide, the inner surface of the base part is orange red, the leaf sheath is rolled back outwards, the upper part is provided with a ridge, and no hair exists or the connection part of the leaf sheath and the leaf is slightly hairy; the middle pulse is thick, the lower part is narrow gradually, the base part is narrower than the leaf sheath, the upper part is provided with micro hair, the tip is long and sharp gradually, the side pulse is smooth, the edge of the leaf is jagged, and the lower part is pink green. The stem and leaf is used as raw material for extracting vanillin (Citronellal) as essential oil. The fresh leaves have high oil content of 0.6-0.7 percent, and the essential oil has the total Geraniol (Geraniol) content of 83-92 percent, is expensive and has high quality. And has good effect in treating epilepsy and anxiety.
Currently, the world is distributed mainly in Guangdong and Hainan China (Xinglong Fushan), Taiwan cultivation, India, Srilanca, Malaysia, Indonesia, and other countries. 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 life quality, the demand of China on the citronella is continuously increased. The citronella serves as a typical tropical characteristic spice crop, has important significance for developing local characteristic economy and improving income and living standard of farmers in remote areas, and has wide development prospect.
The conventional planting method mainly adopts the branch breeding because the flowering and fruiting rate of the citronella is low. The method has large demand on the stock plant, and cannot meet the requirement of the industry on high-throughput seedling breeding, and the further development of the citronella industry 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, a maternal tissue is taken as an explant, a callus is induced under a proper environment, the maternal tissue is directly intercepted by replacing the callus with a large amount 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. This process enables the development of many individuals from one maternal tissue, greatly exciting the reproductive capacity of the tissue of the citronella. However, no tissue rapid propagation research on the citronella is available at present.
Disclosure of Invention
In view of the above, the invention provides a high-throughput breeding method for seedlings of citronella. The method realizes the high-flux breeding of the seedlings of the citronella, enlarges the scale of the seedlings of the citronella and lays a foundation for the genetic transformation breeding in the future.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a high-throughput breeding method of a seedling of a citronella, which comprises the following steps:
removing leaf sheath on the surface of the parent plant of the citronella, taking the base of the stem section which is close to the root end and semi-lignified as an explant, 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 0.1-0.5 mg/L2, 4-D and 0.5-1.5 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 1.0-2.5mg/L of 6-BA and 0.1-0.5mg/L of NAA;
after the cluster buds are divided into plants, the plant buds are inoculated to a proliferation and rooting culture medium for proliferation and rooting to obtain a complete plant; the proliferation and rooting culture medium is 1/2MS culture medium containing 1.0-2.5 mg/L6-BA and 0.1-0.5mg/L NAA.
Preferably, the length of the base of the semi-lignified stem segment near the root end is 3-5 mm.
Preferably, the cleaning is: and washing the explants with a detergent, and washing the explants for 10-30min with running water.
Preferably, the disinfection and sterilization is as follows: transferring the explant to a sterile operation table, disinfecting the surface of the explant by using 75% alcohol for 30-50s, washing the explant by using sterile water for 3-4 times, then sterilizing the explant by using 0.1% mercuric chloride for 5-8min, washing the explant by using the sterile water for 4-5 times, washing the explant by using the sterile water for 1-2min each time, and draining water.
Preferably, the callus induction medium is MS medium containing 0.1 mg/L2, 4-D and 0.5 mg/L6-BA.
Preferably, the callus induction condition is that the callus is cultured for 25-35 days at 28 +/-2 ℃ in the dark.
Preferably, the callus induction conditions are 28 + -2 deg.C in the dark for 30 days.
Preferably, the cluster bud induction medium is MS medium containing 1.0 mg/L6-BA and 0.1mg/L NAA.
Preferably, the condition for inducing the cluster buds is to culture the cluster buds for 25-35 days at the temperature of 28 +/-2 ℃ and the illumination intensity of 1000-2500 lux.
Preferably, the conditions for cluster bud induction are 28. + -. 2 ℃ for 30 days at a light intensity of 2500 lux.
Preferably, the proliferation and rooting culture medium is 1/2MS culture medium containing 1.0 mg/L6-BA and 0.1mg/L NAA.
Preferably, the condition of proliferation and rooting is culturing for 25-35 days at the temperature of 28 +/-2 ℃ and the illumination intensity of 1500-2500 lux.
Preferably, the condition for proliferating and rooting is culturing for 30 days at 28 +/-2 ℃ and illumination intensity of 2500 lux.
The invention provides a high-throughput breeding method of a seedling of a citronella, which comprises the following steps: removing leaf sheath on the surface of the separated plant of the parent of the citronella, taking the base of the stem section which is close to the root end and semi-lignified as an external implant, 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 0.1-0.5 mg/L2, 4-D and 0.5-1.5 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 1.0-2.5mg/L of 6-BA and 0.1-0.5mg/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 0.1-0.5mg/L NAA. The invention has the following beneficial effects:
the method of the invention selects the stem base as the explant, and the callus induction culture medium formula is as follows: adding 2, 4-dichlorophenoxyacetic acid (2,4-D) and 6-benzylamino adenine (6-BA) into 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-benzylamino adenine (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 citronella seedlings.
In the method, the stem base of the citronella is used as a material to obtain callus, and further the regeneration of the clumpy buds is induced to finally obtain a complete plant. The invention takes different explants and culture media as comparison methods, and cultures under the same environmental conditions as the method of the invention, the result shows that only the stem base 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 no callus or cluster buds are obtained, therefore, the method of the invention is obviously superior to the comparison methods in the aspects of callus induction and cluster bud regeneration of the maple grass.
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FIG. 1 shows the explant material, callus, cluster buds and complete plant of the sweetgum grass seedling in the tissue culture and rapid propagation method of the invention; wherein 1-1 is the maple, 1-2 is the stem section of the maple, 1-3 is the base of the stem section of the maple, 1-4 is callus induction, 1-5 is the induction of the clump bud, and 1-6 is the proliferation and rooting of the clump bud.
Detailed Description
The invention discloses a high-throughput breeding method of a citronella seedling, which can be realized by appropriately improving process parameters by a person skilled in the art with the aid of the content in the text. 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 provides a tissue rapid propagation technical method of a seedling of a citronella, which comprises the following steps:
step 1, selection and sterilization of explants: cutting a parent plant, poking leaf sheaths on the upper surface of the parent plant of the maple grass 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 platform, sterilizing the surface of the product with 75% alcohol for 30-50s, washing the product with sterile water for 3-4 times, sterilizing the product with 0.1% mercuric chloride for 5-8min, washing the product with sterile water for 4-5 times, washing the product with sterile water for 1-2min each time, and draining the water for inoculation.
Step 2, explant induced callus: 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 is MS +0.1-0.5 mg/L2, 4-dichlorophenoxyacetic acid (2,4-D) +0.5-1.5 mg/L6-benzylamino adenine (6-BA).
Step 3, inducing and differentiating the cluster buds by the callus: the induced callus is inoculated to a cluster bud induction culture medium in small blocks for cluster bud induction, and the cluster bud induction is carried out at the temperature of 28 +/-2 ℃ and the illumination intensity of 1000-2000lux, wherein the formula of the culture medium is MS +1.0-2.5 mg/L6-benzylamino adenine (6-BA) +0.1-0.5mg/L Naphthyl Acetic Acid (NAA).
Step 4, proliferating and rooting the cluster buds: the induced cluster buds are divided into plants and inoculated to a proliferation and rooting culture medium, the illumination intensity is 1500-.
Preferably, step 1 is:
pulling leaf sheaths on the upper surface of the maple grass stock plant layer by layer until the semi-lignified stem base part close to the root end is exposed, then cutting the semi-lignified stem segment base part with the length of 3-5mm, and inoculating the stem segment base part to a callus induction culture medium after sterilization.
Preferably, the callus induction medium formula is MS-based medium, and comprises 2, 4-dichlorophenoxyacetic acid (2,4-D) and 6-benzylamino adenine (6-BA); MS is taken as a basic culture medium of the cluster bud differentiation culture medium and contains 6-benzylamino adenine (6-BA) and naphthylacetic acid (NAA); the proliferation and rooting culture medium takes 1/2MS as a basic culture medium and comprises 6-benzylamino adenine (6-BA) and Naphthalene Acetic Acid (NAA).
In the specific embodiment of the invention, the stem base of the citronella (Cymbopogon winerianus Jowitt.) is used as a material to obtain callus, and then the regeneration of the clumpy buds is induced, and finally the complete plant is obtained.
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 maple grass.
According to the technical scheme, the stem base is selected as the explant, and the callus induction culture medium formula comprises the following components: adding 2, 4-dichlorophenoxyacetic acid (2,4-D) and 6-benzylamino adenine (6-BA) into 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-benzylamino adenine (6-BA) and naphthylacetic acid (NAA) are added, so that a rhizome section base part can have the differentiation rate of more than 18-25 clustered buds through culture, and the method has important significance for high-throughput breeding and genetic engineering research of future seedlings of the chaulmoogra.
The invention relates to the technical field of tissue culture and high-throughput seedling breeding of sweetgum cudweed, and discloses a seedling rapid propagation technical method taking a stem base of the sweetgum cudweed as an explant. The method comprises the steps of poking leaf sheaths on the upper surface of a maple grass parent plant layer by layer until semi-lignified stem base parts close to root ends are exposed, cutting the semi-lignified stem segment base parts with the length of 3-5mm, and inducing callus after disinfection and sterilization; then differentiating cluster buds by using the callus, further carrying out bud division and proliferation, and finally rooting and growing into complete plants so as to achieve the aim of high-throughput breeding. White callus is generated around the stem base after the first culture for 25 to 35 days on the MS culture medium containing 2,4-D and 6-BA, and then the callus is further proliferated into embryogenic callus and non-embryogenic callus, wherein the embryogenic callus is in a white dispersed particle state, and the non-embryogenic callus is in a brown color and is stacked into a lump. Selecting the embryonic callus to culture on the MS culture medium containing 6-BA and NAA for 14 to 21 days, then the embryonic callus begins to turn green locally and begins to extract cluster buds, after the culture is continued for 25 days, the cluster buds containing 6-BA and NAA in 1/2MS culture medium are further proliferated and differentiated, finally, the cluster buds with more than 18-25 rhizome segments can be separated from one stem base part, and the cluster buds can be regenerated into a complete plant. The in vitro regeneration method of the citronella provides technical support for the high-throughput breeding and the genetic engineering research of the future seedlings of the citronella.
The materials, hormones or culture media used in the tissue rapid propagation method of the citronella provided by the invention can be purchased from the market.
The invention is further illustrated by the following examples:
EXAMPLE 1 Festuca arundinacea propagation method of the present invention
Cutting a parent plant, poking leaf sheaths on the upper surface of the parent plant of the maple grass 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 foam. 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 callus induction culture medium, and performing light-shielding culture at 28 + -2 deg.C for callus induction for 30 days, wherein the culture medium formula is MS +0.1 mg/L2, 4-dichlorophenoxyacetic acid (2,4-D) +0.5 mg/L6-benzylamino adenine (6-BA).
Inoculating the induced callus to a cluster bud induction culture medium in small blocks for cluster bud induction, culturing at 28 + -2 deg.C with illumination intensity of 2500lux for 30 days, wherein the culture medium formula is MS +1.0 mg/L6-benzylamino adenine (6-BA) +0.1mg/L Naphthalene Acetic Acid (NAA).
The induced cluster buds are divided into plants and inoculated to a proliferation and rooting culture medium, the illumination intensity is 2500lux at the temperature of 28 +/-2 ℃, the proliferation and rooting are carried out, the culture is carried out for 30 days, and the formula of the culture medium is 1/2MS +1.0 mg/L6-benzylamino adenine (6-BA) +0.1mg/L naphthylacetic acid (NAA).
In the breeding method, the explant material, the callus, the cluster buds and the complete plant of the curculigo orchioides are shown in figure 1. The black box marks the key sampling site (1-2 in FIG. 1) of the Cure induced by the Curculigo curcas, and the callus can not be induced by other leaves and stem segments. Callus is differentiated from the periphery of the basal part, the differentiation and regeneration are strong, cluster buds can be induced by culturing for 2 weeks, and the whole plant can be regenerated.
Comparative example 1:
in the comparative example, the culture method in example 1 was used with the tender leaves and tender stems of Curculigo orchioides as explants.
Taking tender leaves and tender stems of the Acertruncatum Bunge stock plant 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 leaf on callus induction culture medium, and performing light-shielding culture at 28 + -2 deg.C for callus induction for 30 days, wherein the culture medium formula is MS +0.1 mg/L2, 4-dichlorophenoxyacetic acid (2,4-D) +0.5 mg/L6-benzylamino adenine (6-BA). But no callus was induced.
Comparative example 2:
in this comparative example, 6-BA added to the callus induction medium in example 1 was changed to KT, and the other operation steps were the same.
Cutting a parent plant, poking leaf sheaths on the upper surface of the parent plant of the maple grass 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 foam. 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 for 30 days, 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, culturing at 28 + -2 deg.C with illumination intensity of 2500lux for 30 days, wherein the culture medium formula 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, the illumination intensity is 2500lux at the temperature of 28 +/-2 ℃, the proliferation and rooting are carried out, the culture is carried out for 30 days, 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:
in this comparative example, young leaves and young stems were used as explant materials, 6-BA added to the callus induction medium in example 1 was changed to KT, and the other operation steps were the same as in example 1.
Taking tender leaves and tender stems of the Acertruncatum Bunge stock plant 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 leaf on callus induction culture medium, and performing light-shielding culture at 28 + -2 deg.C for callus induction for 30 days, wherein the culture medium formula is MS +0.1 mg/L2, 4-dichlorophenoxyacetic acid (2,4-D) +0.5mg/L Kinetin (KT). But no callus was induced.
Test example:
the test was conducted according to the methods of example 1 and comparative examples 1 to 3, and the induction rate of callus induction for 30 days and the induction rate of cluster buds induction for 30 days 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 stem segment base as the explant is more than 20 times. By adopting the culture method of callus induction and cluster bud differentiation provided by the invention, the callus induction rate reaches 72%, and the cluster bud differentiation rate reaches about 80%, 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 (4)
1. A high-throughput breeding method of a seedling of a citronella is characterized by comprising the following steps:
removing leaf sheath on the surface of the parent plant of the citronella, taking the base of the stem section which is close to the root end and semi-lignified as an explant, 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 0.1 mg/L2, 4-D and 0.5 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 1.0mg/L of 6-BA and 0.1mg/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 mg/L6-BA and 0.1mg/L NAA;
the callus induction condition is that the callus is cultured for 25-35 days in the dark at the temperature of 28 +/-2 ℃;
the condition for inducing the cluster buds is that the cluster buds are cultured for 25-30 days at the illumination intensity of 1000-2500 lux at the temperature of 28 +/-2 ℃;
the condition of proliferation and rooting is that the culture is carried out for 25-35 days at the temperature of 28 +/-2 ℃ and the illumination intensity of 1500-2500 lux.
2. The high-throughput breeding method of claim 1, wherein the length of the base of the stem segment near the root end and semi-lignified is 3-5 mm.
3. The high-throughput breeding method according to claim 1, wherein the washing is: and washing the explants with a detergent, and washing the explants for 10-30min with running water.
4. The high-throughput breeding method according to claim 1, wherein the sterilization is: transferring the explant to a sterile operation table, carrying out surface sterilization on the explant by using 75% alcohol for 30-50s, washing the explant by using sterile water for 3-4 times, then carrying out sterilization by using 0.1% mercuric chloride for 5-8min, washing the explant by using the sterile water for 4-5 times, washing the explant by using the sterile water for 1-2min each time, and draining water.
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