CN110506632B - Tissue culture method of euscaphis konishii hayata - Google Patents
Tissue culture method of euscaphis konishii hayata Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
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Abstract
A tissue culture method of euscaphis konishii hayata relates to a plant tissue culture method. Selecting the multiple leaves of the yellow-fruit euscaphis konishii hayata as an explant, setting a buffer experiment mode of 5-7d at the early stage of bottle seedling induction by using the physiological habit of the multiple leaves of the yellow-fruit euscaphis konishii hayata, selecting to obtain the sterile explant, gradually dropping off the small leaves in the culture process, forming wound surfaces on leaf shafts, further culturing callus tissues, obtaining the regenerated plant of the yellow-fruit euscaphis konishii hayata, and well solving the technical problem of tissue culture of the yellow-fruit euscaphis konishii hayata. The euscaphis konishii hayata compound leaves are used as explants, the processes of primary induction, callus induction, seedling induction, cluster bud induction, rooting induction and domestication culture are all materials required by conventional tissue culture, a culture medium is easy to obtain, equipment is simple, and the cost is low.
Description
Technical Field
The invention relates to a plant tissue culture method, in particular to a tissue culture method of euscaphis konishii hayata.
Background
Euscaphis japonica (Thunberg) Kanitz is evergreen or deciduous small arbor or shrub of Euscaphis japonica of Staphyleaceae, namely Euscaphis japonica (Staphyleaceae), has whole stem fruits and bright red mature fruit peels, and has high ornamental and medicinal values. Euscaphis japonica (Thunberg) Kanitzvar. wupingensis B.P.Cai et Z.R.Chen) is a natural variant in the geographical distribution area of Fujian province, and was formally published in 2018 by Zea Bang et al (Bang-Ping Cai, etc.2018.Euscaphis japonica var. wupingensis, a new variety with yellow pigment from Fujian, China. Phytotaxa,334(1):055 + 059). The annual branches are yellow green, the leaves are in odd feathery multiple leaves, the leaves are opposite, the leaf stalks are longer and softer than the euscaphis konishii hayata, the stem uprightness is inferior to the euscaphis konishii hayata, the mature endocarp of the euscaphis konishii hayata is yellow, the eversion is butterfly wing, and the fleshy aril is bright black; the fruit bearing period can be from 9 months to about 3 months in the next year, and the fruit bearing agent has great ornamental, medicinal and research values.
Most of the traditional euscaphis konishii hayata cultivation modes are seed propagation, the euscaphis konishii hayata has the characteristics of hard seed coat and deep dormancy of seeds, the period from sowing to emergence lasts for nearly 3 years, and the emergence rate is low. The yellow fruit euscaphis konishii hayata is a natural variation species of the euscaphis konishii hayata discovered in recent years, the natural distribution area is narrow, germplasm resources are rare, the fruit is rich in nutrition and is easy to eat by birds and animals, plants dug by human beings are used as medicinal and ornamental tree species, the number of the plants in a natural habitat is small, the natural fruiting rate is low, the hereditary character is influenced by various factors such as environment and pollination, and once the yellow fruit euscaphis konishii hayata is discovered, the yellow fruit euscaphis konishii hayata becomes a rare or endanger. In addition, the problems of recalcitrant seeds, irregular seedling emergence, long cultivation period and the like exist, and the propagation coefficient of the seeds of the euscaphis konishii hayata is very low. The fruit of the euscaphis japonica is yellow, so that the euscaphis japonica is obviously different from euscaphis japonica and has great demand on the market. The single seed culture and propagation of the seedling can not meet the market demand.
Disclosure of Invention
The discovery aims at providing a tissue culture method of euscaphis konishii hayata for obtaining asexual regeneration plants by using compound leaves with leaves of euscaphis konishii hayata as explants through a tissue culture technology.
The invention comprises the following steps:
the method includes the steps of intercepting branch-free branches of annual yellow fruit crow tree toon, and performing indoor water planting after washing;
selecting blades close to the top ends of the branches, cleaning, washing, draining water, and putting into a superclean workbench; cutting compound leaves, soaking the compound leaves in 75% ethanol, washing with sterile water, sterilizing with mercury solution, washing with sterile water, and draining to obtain small segments of the compound leaves of euscaphis konishii hayata for later use;
inoculating the euscaphis konishii hayata compound leaf small section obtained in the step II into an MS hormone-free culture medium for sterile induction culture for 5-7 days to obtain a sterile euscaphis konishii hayata compound leaf small section;
taking out the sterile euscaphis konishii hayata multiple-leaf small sections obtained in the step three from a culture medium, reserving about 0.5cm on a super-clean workbench along the leaf axis direction, cutting off the rest small leaves, inoculating the small leaves into a callus induction culture medium for callus induction, and culturing for 120-150 days to differentiate a certain number of euscaphis konishii hayata callus;
fifthly, transferring the calli of the yellow fruit euscaphis sinensis obtained in the step four to a primary induction culture medium for seedling induction, and culturing for emergence for about 30 days to obtain yellow fruit euscaphis sinensis seedlings;
sixthly, transferring the yellow fruit euscaphis sinensis seedlings obtained in the step of transferring the yellow fruit euscaphis sinensis seedlings into a multiplication culture medium, and performing cluster bud induction for about 30 days to obtain the yellow fruit euscaphis;
selecting 2-4 multi-leaf plants with the plant height of more than 1cm and strong growth from the euscaphis konishii hayata seedlings obtained in the step VI, and transferring the plants to a rooting culture medium for rooting induction, wherein the culture period is 30-60 d;
and selecting strong seedlings with the plant height of 2-3 cm, 6 growing multiple leaves and the root number of 3-5 from the yellow-fruit euscaphis konishii hayata seedlings subjected to root induction, carrying out outdoor acclimation culture with the seedlings in bottles, uncovering, shading and moisturizing, wherein the culture period is 5-7 d.
In the step, intercepting the branch-free branches of annual yellow fruit crow tree, and the specific steps of washing indoor water culture can be as follows: intercepting branches of annual yellow fruit euscaphis konishii hayata without branches, washing for 3-5 min under tap water, carrying out indoor water culture for 5-7d, replacing clear water once every day, and selecting leaves close to the top ends of the branches.
In the second step, the leaves close to the top ends of the branches are selected for cleaning, the leaves with the stems close to the top ends of the branches can be selected for washing by adding detergent, and the leaves are washed for 10-15 min in tap water; the specific method for washing the cut compound leaves with sterile water after being soaked in 75% ethanol comprises the following steps: cutting compound leaves, reserving leaf axis and 2-3 complete small leaves at each section, cutting short at the upper part and long at the lower part according to the growth direction, soaking for 30s by using 75% ethanol, and washing by using sterile water; the mercury solution can be sterilized by 0.1% mercuric chloride solution for 4-8 min.
In the step three, the MS hormone-free culture medium is: 1/2MS + 20-25 g/L sucrose + 7-8 g/L agar, and pH5.8-6.2.
In step four, the callus induction medium is: 1/2MS + 20-25 g/L sucrose + 7-8 g/L agar, and pH 5.8-6.2.
In step fifthly, the callus of the yellow fruit euscaphis konishii hayata is preferably compact in texture and slightly green tissue blocks; the primary induction culture medium comprises: 4/5MS +6-BA 0.5-1.0 mg/L + NAA 0.05-0.1 mg/L + sucrose + 7-8 g/L agar, pH 5.8-6.2.
In the step sixteenth, the proliferation medium is: 1.0-1.5 mg/L of MS +6-BA + 0.1-0.2 mg/L of NAA + 25-30 g/L of sucrose + 8-9 g/L of agar, and the pH value is 5.8-6.2.
In step-quietness, the rooting medium is: 1/2WPS + NAA 1.0-2.0 mg/L + 2.4-D1.0-2.0 mg/L + 20-25 g/L sucrose + 8-9 g/L agar + 2-3 g/L activated carbon, and pH 5.8-6.2.
The invention has the innovation points that sterile explants are obtained by selecting the compound leaves of the euscaphis konishii hayata as explants and setting a buffer experiment mode of 5-7d at the early stage of the induction of the vase seedlings by using the physiological habit of the compound leaves of the euscaphis konishii hayata, small leaves gradually fall off in the culture process, wound surfaces are formed on leaf shafts, then callus tissues are cultured, regenerated plants of the euscaphis konishii hayata are obtained, and the technical problem of tissue culture of the euscaphis konishii hayata is well solved.
Compared with the prior art, the invention has the following outstanding advantages:
easy realizability: the invention uses the compound leaves with the petioles of the euscaphis konishii hayata as explants, and can form regeneration plants by proper tissue culture technology.
The sterility and high efficiency are as follows: according to the invention, sterile explants are selected by setting a 5-7d buffer experiment mode at the early induction stage of the bottle seedling through the physiological habit of the compound leaves of the yellow fruit euscaphis konishii hayata, small leaves gradually fall off in the culture process, wound surfaces are formed on leaf shafts, and then callus tissues are cultured, so that the aim of plant regeneration of the yellow fruit euscaphis konishii hayata is fulfilled.
The production cost is low: the invention takes the yellow fruit euscaphis konishii hayata compound leaves as explants, the processes of primary induction, callus induction, seedling induction, cluster bud induction, rooting induction and domestication culture are all materials required by conventional tissue culture, the culture medium is easy to obtain, the equipment is simple, and the cost is low.
Drawings
FIG. 1 is a photograph of the induction culture of the leaf axis of Euscaphis konishii.
FIG. 2 is a photograph of a seedling induction experiment of Euscaphis konishii. The marker H is yellow fruit euscaphis konishii hayata, and 4.20 is induction date.
FIG. 3 is a photograph showing the cultivation of clustered shoots of Euscaphis konishii.
FIG. 4 is a photograph of rooting culture of euscaphis konishii hayata.
FIG. 5 is a photograph showing the fungal infection of explants of stem segments of Euscaphis konishii (comparative example).
FIG. 6 is a photograph showing the fungal infection of the stem and leaf explants of Euscaphis konishii hayata (comparative example).
Detailed Description
The following examples will further illustrate the present invention with reference to the accompanying drawings.
The embodiment of the invention comprises the following steps:
the selection and treatment method of the explant comprises the following steps: selecting branches of annual yellow fruit euscaphis konishii hayata without branches, washing the branches for 3-5 min under tap water, carrying out indoor water culture for 5-7d, and replacing clear water every day for purification; then, selecting the leaves with the petioles, which are close to the top ends of the branches, adding detergent for cleaning and disinfection, washing for 10-15 min under tap water, draining water, and placing the leaves into an ultra-clean workbench; cutting compound leaves, wherein each section is provided with a leaf axis and 2-3 complete small leaves, and the upper part of each section is truncated and the lower part of each section is truncated according to the growth direction; taking the cut compound leaves as explants, soaking the explants in 75% ethanol for 30s, washing the explants with sterile water, sterilizing the explants with 0.1% mercuric chloride solution for 4-8 min, washing the explants with sterile water, and draining the water for later use.
The primary induction method comprises the following steps: and inoculating the euscaphis konishii hayata compound leaf small segments to a hormone-free culture medium 1/2MS + 20-25 g/L sucrose + 7-8 g/L agar, and carrying out sterile induction culture at a pH of 5.8-6.2 for 5-7 days.
The callus induction method comprises the following steps: leaving a small section of sterile euscaphis konishii hayata with about 0.5cm along the direction of a leaf axis, cutting off the rest small leaves, inoculating the small section to a culture medium 1/2MS + 20-25 g/L sucrose + 7-8 g/L agar, culturing at the pH of 5.8-6.2 for 120-150 days;
the seedling induction method comprises the following steps: transferring the compact and slightly green tissue blocks to a culture medium 4/5MS +6-BA 0.5-1.0 mg/L + NAA 0.05-0.1 mg/L + 20-25 g/L sucrose + 7-8 g/L agar, and culturing for about 30 days to obtain seedlings, wherein the pH value is 5.8-6.2;
the cluster bud induction method comprises the following steps: the euscaphis konishii hayata seedlings are inoculated to a culture medium MS +6-BA 1.0-1.5 mg/L + NAA 0.1-0.2 mg/L + 25-30 g/L sucrose + 8-9 g/L agar, the pH value is 5.8-6.2, and the culture lasts for about 30 days;
the rooting induction method comprises the following steps: selecting yellow fruit euscaphis konishii hayata seedlings with the height of more than 1cm, growing 2-4 compound leaves and growing robustly, inoculating the single plants to a culture medium 1/2WPS + NAA 1.0-2.0 mg/L + 2.4-D1.0-2.0 mg/L + 20-25 g/L sucrose + 8-9 g/L agar + 2-3 g/L active carbon, and culturing for 30-60 days at the pH of 5.8-6.2;
the domestication culture method comprises the following steps: selecting strong yellow fruit euscaphis konishii hayata seedlings with the plant height of 2-3 cm, 6 growing leaves and the number of 3-5, wherein the root length is more than 2cm, and the strong seedlings grow, carrying out outdoor domestication with bottles, covers and shades, and keeping the moisture culture period for 5-7 days.
The indoor growth environmental conditions of the bottle seedlings are as follows: the room temperature is 26 +/-2 ℃, the illumination intensity is 1500-2500 Lux, and the illumination culture is 12 hr/d.
Specific examples are given below.
Example (b):
the embodiment of the invention comprises explant selection and treatment and tissue culture.
Explant selection and processing: selecting an annual yellow-fruit euscaphis konishii hayata which has no plant diseases and insect pests and is robust, obliquely cutting the cross section, washing for 3-5 min under tap water, moving to indoor water culture for 5-7d, and replacing clear water every day for once purification; selecting leaves with leaves and stems close to the top ends of branches after 5 days of indoor water culture, adding a detergent for cleaning, washing for 10-15 min under tap water, draining water, and placing the leaves on a super-clean workbench; cutting compound leaves, wherein each section is provided with a leaf axis and 2-3 complete small leaves, and the upper part of each section is truncated and the lower part of each section is truncated according to the growth direction; taking cut compound leaves as explants, soaking the cut compound leaves in 75% ethanol for 30s, washing the explant with sterile water, sterilizing the explant with 0.1% mercuric chloride solution for 4-8 min, washing the explant with sterile water, and draining off water for later use;
the tissue culture comprises a technical system of primary induction, callus induction, seedling induction, cluster bud induction, rooting induction and acclimatization culture.
Primary induction: inoculating the euscaphis konishii hayata multiple-leaf small segments to a hormone-free culture medium 1/2MS +25g/L sucrose +8g/L agar, and carrying out sterile induction culture at the pH of 5.8-6.0 for 5-7d, wherein the sterile induction survival rate is about 5%;
callus induction: leaving a small section of sterile euscaphis konishii hayata multiple leaves about 0.5cm along the direction of a leaf axis, cutting off the rest small leaves, inoculating the small section to a culture medium 1/2MS +25g/L sucrose +8g/L agar, culturing for about 150 days at a pH value of 5.8-6.2, and allowing the diameter of callus around the leaf axis to reach about 0.5 cm;
seedling induction: transferring the compact and slightly green tissue blocks to a culture medium 4/5MS +6-BA 0.5-1.0 mg/L + NAA
0.05-0.1 mg/L + 20-25 g/L sucrose + 7-8 g/L agar, pH5.8-6.2, and culturing for about 30 days to emerge;
inducing cluster buds: yellow fruit euscaphis konishii hayata seedlings are inoculated in a culture medium MS +6-BA 1.0-1.5 mg/L + NAA 0.1-0.2 mg/L
+25g/L of sucrose and 9g/L of agar, the pH value is 5.8-6.2, the culture lasts for about 30 days, and the effective multiplication coefficient is more than 8.0;
rooting induction: selecting yellow fruit euscaphis konishii hayata seedlings with the height of more than 1cm, growing 2-4 compound leaves and growing robustly, inoculating the single plants in a culture medium 1/2WPS + NAA 1.0mg/L + 2.4-D2.0 mg/L +20g/L sucrose +9g/L agar +2g/L active carbon with the pH value of 5.8-6.2, and culturing for about 60 days, wherein the rooting rate reaches 93.3%;
domestication and culture: selecting euscaphis konishii hayata seedlings with the height of 2-3 cm, 6 growing leaves and the number of 3-5, the root length of more than 2cm and strong seedlings, carrying out outdoor domestication with bottles, covers and shades, wherein the period of moisturizing culture is 5-7d, and the survival rate reaches 84%.
Comparative example 1:
explant selection and processing: selecting an annual yellow-fruit euscaphis konishii hayata which has no plant diseases and insect pests and is robust, obliquely cutting the cross section, washing for 3-5 min under tap water, moving to indoor water culture for 5-7d, and replacing clear water once every day; selecting stem sections with axillary buds on branches subjected to indoor water culture for 5 days, adding detergent for cleaning, washing for 15-20 min under tap water, draining water, and placing the branches on an ultra-clean workbench; soaking in 75% ethanol for 30s, washing with sterile water, sterilizing with 0.1% mercuric chloride solution for 8-12 min, washing with sterile water, and draining off water;
primary induction: inoculating stem segments with axillary buds of the yellow-fruit euscaphis konishii hayata to a hormone-free culture medium 1/2MS +25g/L sucrose +8g/L agar, and carrying out sterile induction culture at the pH of 5.8-6.0 for 3-5 days, wherein hyphae appear on the section of the stem segments, the growth speed of the hyphae is superior to the germination speed of explants, and the hyphae are spread around the explants in about 7 days; the explants die due to browning after 7-10 days.
Comparative example 2:
explant selection and processing: selecting an annual yellow-fruit euscaphis konishii hayata which has no plant diseases and insect pests and is robust, obliquely cutting the cross section, washing for 3-5 min under tap water, moving to indoor water culture for 5-7d, and replacing clear water once every day; selecting leaves close to the top end of a branch on the branch subjected to indoor water culture for 5 days, adding a detergent for cleaning, flushing for 10-15 min under tap water, draining water, and placing the branch on an ultra-clean workbench; soaking in 75% ethanol for 30s, washing with sterile water, sterilizing with 0.1% mercuric chloride solution for 4-8 min, washing with sterile water, and draining off water;
primary induction: inoculating the double-leaf leaflets of euscaphis konishii hayata to a hormone-free culture medium 1/2MS +25g/L sucrose +8g/L agar, and carrying out sterile induction culture at the pH of 5.8-6.0 for 3-5 days, wherein white hyphae appear on the cut surfaces of the leaflets, and the hyphae spread around the explant for about 7 days; the explants die due to browning after 7-10 days.
FIG. 1 shows the photo of the induction culture of the leaf axis of Euscaphis konishii. The multiple leaf axis of the euscaphis konishii hayata explant can be expanded to form callus. FIG. 2 shows the photographs of the induction experiment of the seedlings of euscaphis konishii hayata. FIG. 3 shows photographs of the clustered shoot culture of Euscaphis konishii. FIG. 4 shows photographs of rooting culture of euscaphis konishii hayata. FIG. 5 shows photographs of fungal infections of explants of stem segments of Euscaphis konishii (comparative example). FIG. 6 shows photographs of fungal infections of stem segments and leaf explants of Euscaphis konishii (comparative example).
As can be seen from the examples and the comparative examples, the stem segment with axillary buds or the compound leaf lobule of euscaphis konishii hayata is taken as the explant material to be subjected to sterile induction culture, but mycelium appears on the section of the explant within about 7 days, and the explant dies due to browning after 7-10 days; in the embodiment, in the process of sterile induction culture by taking compound leaves with petioles as explants, sterile seedlings are screened for 5-7d and transferred again to induce the callus formation of the explants, so that the phenomenon that the explants are browned and die due to fungus infection in the induction process is avoided.
The plant tissue culture mode can rapidly propagate the asexual plant. The tissue culture method of euscaphis konishii hayata has been a breakthrough in recent years and has a mature technology. In the experimental research process, the traditional euscaphis konishii hayata explant material taking mode is adopted, and sterile induction is successively carried out on stem sections, axillary buds, leaves and the like of euscaphis konishii hayata, and the sterile induction is not successful.
Through multiple experimental researches, the regenerated plant of the euscaphis konishii hayata is successfully induced and obtained by using the compound leaves with leaves of the euscaphis konishii hayata as the explant, and a tissue culture system of the euscaphis konishii hayata is established. The invention lays an important foundation for tissue culture and industrial seedling culture of euscaphis konishii hayata in future.
Claims (5)
1. A tissue culture method of euscaphis konishii hayata is characterized by comprising the following steps:
the method includes the steps of intercepting branch-free branches of annual yellow fruit crow tree toon, and performing indoor water planting after washing;
selecting blades close to the top ends of the branches, cleaning, washing, draining water, and putting into a superclean workbench; cutting compound leaves, soaking the compound leaves in 75% ethanol, washing with sterile water, sterilizing with mercury solution, washing with sterile water, and draining to obtain small segments of the compound leaves of euscaphis konishii hayata for later use;
inoculating the euscaphis konishii hayata compound leaf small section obtained in the step II into an MS hormone-free culture medium for sterile induction culture for 5-7 days to obtain a sterile euscaphis konishii hayata compound leaf small section; the MS hormone-free culture medium comprises: 1/2MS + 20-25 g/L sucrose + 7-8 g/L agar, pH5.8-6.2;
taking out the sterile euscaphis konishii hayata multiple-leaf small sections obtained in the step three from a culture medium, reserving 0.5cm on a super-clean workbench along the leaf axis direction, cutting off the rest small leaves, inoculating the small leaves into a callus induction culture medium for callus induction, and culturing for 120-150 days to differentiate a certain number of euscaphis konishii hayata callus; the callus induction culture medium comprises: 1/2MS + 20-25 g/L sucrose + 7-8 g/L agar, and pH is 5.8-6.2;
fifthly, transferring the calli of the yellow fruit euscaphis sinensis obtained in the step four to a primary induction culture medium for seedling induction, and performing culture for 30d to obtain seedlings of the yellow fruit euscaphis sinensis; the primary induction culture medium comprises: 4/5MS +6-BA 0.5-1.0 mg/L + NAA 0.05-0.1 mg/L + sucrose + 7-8 g/L agar, pH 5.8-6.2;
sixthly, transferring the yellow fruit euscaphis sinensis seedlings obtained in the step of transferring the yellow fruit euscaphis sinensis seedlings into a multiplication culture medium, and performing cluster bud induction for 30 days to obtain the yellow fruit euscaphis sinensis seedlings; the proliferation culture medium is as follows: 1.0-1.5 mg/L of MS +6-BA + 0.1-0.2 mg/L of NAA + 25-30 g/L of sucrose + 8-9 g/L of agar, and the pH value is 5.8-6.2;
selecting 2-4 multi-leaf plants with the plant height of more than 1cm and strong growth from the euscaphis konishii hayata seedlings obtained in the step VI, and transferring the plants to a rooting culture medium for rooting induction, wherein the culture period is 30-60 d; the rooting culture medium comprises: 1/2 WPM + NAA 1.0-2.0 mg/L +2, 4-D1.0-2.0 mg/L + 20-25 g/L sucrose + 8-9 g/L agar + 2-3 g/L active carbon, pH 5.8-6.2;
and selecting strong seedlings with the plant height of 2-3 cm, 6 growing multiple leaves and the root number of 3-5 from the yellow-fruit euscaphis konishii hayata seedlings subjected to root induction, carrying out outdoor acclimation culture with the seedlings in bottles, uncovering, shading and moisturizing, wherein the culture period is 5-7 d.
2. The tissue culture method of euscaphis konishii hayata according to claim 1, characterized in that in step mess, no branch branches of annual euscaphis konishii hayata are intercepted, and the specific steps of indoor water culture after washing are as follows: intercepting branches of annual yellow fruit euscaphis konishii hayata without branches, washing for 3-5 min under tap water, carrying out indoor water culture for 5-7d, replacing clear water once every day, and selecting leaves close to the top ends of the branches.
3. The tissue culture method of euscaphis konishii hayata according to claim 1, wherein in the second step, the leaves close to the top ends of the branches are selected for cleaning and washing, and the leaves with the petioles close to the top ends of the branches are selected for washing with detergent and washed with tap water for 10-15 min.
4. The tissue culture method of euscaphis konishii hayata as claimed in claim 1, wherein in the step II, the specific method for washing the cut compound leaves with sterile water after being soaked in 75% ethanol comprises the following steps: cutting compound leaves, reserving leaf axis and 2-3 complete small leaves at each section, cutting short at the upper part and long at the lower part according to the growth direction, soaking for 30s by using 75% ethanol, and washing by using sterile water; and (3) sterilizing the mercury solution for 4-8 min by using 0.1% mercuric chloride solution.
5. The tissue culture method of yellow-fruit wild ailanthus altissima as claimed in claim 1, wherein in step fifthly, the yellow-fruit wild ailanthus altissima callus is compact-texture and slightly green tissue blocks.
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