CN111670807B - Tissue culture method of euonymus alatus - Google Patents
Tissue culture method of euonymus alatus Download PDFInfo
- Publication number
- CN111670807B CN111670807B CN202010342971.XA CN202010342971A CN111670807B CN 111670807 B CN111670807 B CN 111670807B CN 202010342971 A CN202010342971 A CN 202010342971A CN 111670807 B CN111670807 B CN 111670807B
- Authority
- CN
- China
- Prior art keywords
- culture
- callus
- seedlings
- days
- illumination
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- 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
- A01H4/001—Culture apparatus for tissue culture
-
- 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
- A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
-
- 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
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Developmental Biology & Embryology (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- Environmental Sciences (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention discloses a euonymus japonicus leaf as an explant, which is systematically improved from aspects of disinfection of the explant, induction of callus, differentiation culture of the callus, strong seedling culture of euonymus japonicus buds, rooting culture of seedlings and hardening and transplanting of adventitious root seedlings, and provides technical support for cultivating euonymus japonicus seedlings which are not limited by seasons, short in production period and large in propagation coefficient, and meets the increasing demand of the market on euonymus japonicus.
Description
Technical Field
The invention relates to a culture method of plant seedlings, in particular to a culture method of euonymus alatus seedlings.
Background
Euonymus fortunei cv. gold, genus Euonymus, family Euonymus, genus evergreen broadleaf bush. The plants mainly grow under the inter-mountain miscellaneous trees, at forest edges or in the brush, and are distributed to Jilin in various provinces and downstream of the Yangtze river, and cultivation is carried out in various regions. The winglets of euonymus japonicus are peculiar, the leaves are golden in the growing season, the leaves turn red in the resting season, the plant height is 30-50cm, the crown width is 60-80cm, the plant crown is low, the stem sprouting capacity is strong, and the management is extensive. The euonymus alatus has tall and straight tree posture, compact plant type, faster growth and building and shaping resistance; meanwhile, the leaf is golden and does not fade in four seasons, and after the fruit tree is in autumn, the fruit tree is full of fruits, so that the ripe fruits crack in winter, red aril is exposed, and the fruits cannot fall in winter. The bunch of red fruits is embedded in the golden leaf cluster, and the golden leaf red fruits have extremely high ornamental value. On the other hand, the euonymus alatus has the advantages of cold resistance, drought resistance, easy survival, strong resistance to various toxic gases, capability of absorbing and purifying air, smoke resistance and dust absorption. Therefore, the garden is mainly used as hedgerow and boundary-increasing tree, which is not only suitable for courtyard, corridor and the periphery of buildings, but also suitable for the green belt of the trunk road. In the greening of urban courtyards, the plants can be planted in an isolated way, in a row way or in a group way. It can also be used as ornamental plant in the house.
At present, the tissue culture propagation of euonymus plants is reported more, but the literature on the tissue culture propagation of euonymus japonicus is less, and the euonymus japonicus is mainly used for garden greening and can also be planted beside rockery for matching planting or ground cover planting and patterned flower beds. Along with the increasing demand of people on the euonymus alatus, the reproduction of the euonymus alatus is more and more emphasized, and the problem of producing euonymus alatus seedlings with stable characters and good quality at present needs to be solved urgently.
Disclosure of Invention
The purpose of the invention is as follows: the invention aims to provide a culture method of euonymus alatus seedlings, which can culture the euonymus alatus seedlings with no limitation of seasons, short production period and large propagation coefficient.
The technical scheme is as follows: the invention provides a culture method of euonymus alatus seedlings, which comprises the following steps:
(1) tissue culture: selecting leaves at the top of a euonymus alatus plant, and sterilizing the explant by sequentially using alcohol, NaClO solution and mercury bichloride to obtain a sterilized explant;
(2) induction of callus: inoculating the sterilized explants obtained in the step (1) on a callus culture medium for culture to obtain callus;
(3) differentiation culture of callus: cutting the callus in the step (2) into small pieces, and inoculating the small pieces into differential culture to perform differential culture to obtain euonymus alatus buds;
(4) strong seedling culture: cutting the euonymus alatus buds obtained in the step (3) from a differentiation culture medium, inoculating the euonymus alatus buds on a strong seedling culture medium, and performing strong seedling culture to obtain euonymus alatus rootless seedlings;
(5) rooting culture: segmenting the euonymus alatus rootless seedlings obtained in the step (4), and transferring the euonymus alatus rootless seedlings to a rooting culture medium for culture to obtain seedlings with adventitious roots;
(6) hardening and transplanting seedlings: and (5) planting the seedlings with the adventitious roots obtained in the step (5) into a transition seedbed, irrigating and disinfecting, and culturing euonymus alatus seedlings.
Preferably, the alcohol used in the step (1) is alcohol with the volume concentration of 75-80%, and the alcohol is soaked for 5-10 s and then washed by water; the NaClO solution is sterilized by using a NaClO solution with the mass concentration of 10-12% for 10-15 min and then washed by water; the mercuric chloride disinfection is carried out for 5-8 min by using mercuric chloride with the mass concentration of 0.1-0.2%.
Further, before the disinfection, the method also comprises the following steps: repeatedly washing the explant under running water for 30-40min, adding a proper amount of detergent, soaking for 30-40min while oscillating, and moving to a super clean workbench for next disinfection treatment after washing.
Preferably, after the disinfection with alcohol in the step (1), the explants are repeatedly washed and shaken with sterile water for at least 3 times, wherein the washing time is not less than 5min each time, and then disinfected with sodium hypochlorite solution. Similarly, after sterilization with NaClO solution, the explant is repeatedly washed with sterile water for at least 3 times (not less than 5min each time), and then mercuric chloride (HgCl) is added2Solution) sterilization.
Further, the formula of the callus culture medium in the step (2) is as follows: MS, 0.2-0.5mg/L BA, 1.0-2.0 mg/L IAA, 50-100 g/L banana puree, 20-30g/L sucrose and 6.5-7g/L agar, wherein the pH value is 5.8-6.0, the culture temperature is 20-24 ℃, and the culture time is 50-55 days; the culture medium for differential culture is sterile culture in a dark environment.
Preferably, before the callus induction in the step (2), the method further comprises the steps of repeatedly washing the sterilized explant with sterile water, sucking water on the surface of the explant with sterile paper, placing the explant in a culture dish, cutting off the periphery of the explant, vertically cutting 1-2 blades on the main vein of the leaf, and then inoculating the cut main vein on the callus culture medium in the step (2).
Preferably, the length of the small blocks in step (3) is 2-4 mm.
Preferably, the culture medium for the differentiation culture in the step (3) is MS, 1.0-2.0 mg/L BA, 0.1-0.5 mg/L BR, 0.1-0.2mg/L IAA, 50-100 g/L banana puree, 20-30g/L sucrose and 6.5-7g/L agar, the pH value is 5.8-6.0, the culture temperature is 20-24 ℃, the culture time is 25-30 days, and the culture times are 3-4 times of differentiation subculture; the differential culture is illumination culture, the illumination intensity of the illumination culture is 1500-2000 lx, and the illumination time is 10-12 h every day.
Further, the culture medium for strong seedling culture in the step (4) comprises MS, 2.0-3.0mg/L BA, 0.5-1.0mg/L BR, 0.1-0.2mg/L IAA, 100-150g/L banana mud, 20-30g/L sucrose and 7.0-7.5g/L agar, the pH value is 5.8-6.0, the culture temperature is 23-27 ℃, and the culture time is 30-35 days; the culture is aseptic culture under the illumination condition, the illumination intensity of the illumination culture is 2000-2500 lx, and the illumination time is 12-15 h every day.
Preferably, the rooting culture medium in the step (5) is 1/2MS, 0.05-0.1 mg/L BR, 0.5-1.0mg/L IAA, 28-30 g/L sucrose and 7-8 g/L agar, the pH value is 5.8-6.0, the culture temperature is 23-27 ℃, and the culture time is 50-60 days; the culture is aseptic culture under the illumination condition, the illumination intensity of the illumination culture is 2000-2500 lx, and the illumination time is 12-15 h every day.
Further, placing the seeds in the step (6) at the temperature of 18-22 ℃ for 7-10 days before entering the transition seedbed.
Preferably, the placement is in an environment with air ingress.
Further, in the transplanting, the seedling with adventitious roots is taken out of the rooting culture medium, the solid culture medium at the roots of the euonymus alatus tissue culture seedling is cleaned by clear water, and meanwhile, the root damage is reduced as much as possible.
Preferably, the culture medium of the transition seedbed in the step (6) is a mixture of sandy soil, peat soil and vermiculite, the volume ratio of which is (3-4): (2-4): 1-2) in sequence, the cultivation humidity in the transition seedbed is 83-87%, and the cultivation temperature is 21-25 ℃.
Further, the disinfection in the step (6) is implemented by diluting 1000-1500 times of liquid medicine with 75-80% of chlorothalonil wettable powder and continuously spraying for 3-4 times; the disinfection frequency is once every 14 to 17 days.
Further, shading 7-10 days after the transition seedbed in the step (6) is cultivated, and ventilating and removing the covering after the transition seedbed is cultivated in a closed space for 8-12 days.
Has the advantages that: the euonymus alatus seedlings with stable characters can be obtained by the technical means of tissue culture, and the tissue culture has the advantages of no limitation of seasons, short production period, large propagation coefficient and the like. The research uses leaves of euonymus alatus as explants, and systematic research is carried out from aspects of disinfection of the explants, induction of callus, differentiation culture of the callus, strong seedling culture of seedlings, rooting culture of seedlings and the like so as to provide technical support for rapid propagation of excellent euonymus alatus seedlings and meet the increasing demands of markets on euonymus alatus.
Detailed Description
Example 1
1. Sterilizing and disinfecting
Selecting robust seedling plants growing for three years, and selecting the 2 nd leaf of the plant from the top of the plant as an explant.
Repeatedly washing the explant under running water for 30min, adding a proper amount of detergent, soaking for 30min while oscillating, washing, and transferring to a clean bench for next disinfection treatment. The disinfection treatment comprises the following steps: soaking the explant in 75% alcohol for 10s, and repeatedly washing the explant with sterile water for 3 times, wherein the washing time is 5min each time; then transferring the explant into NaClO solution with the mass concentration of 10% for disinfection for 15min, and repeatedly washing and oscillating the explant for 3 times by using sterile water, wherein the washing time is 5min each time; finally, mercury mercuric chloride (HgCl) with the mass concentration of 0.2 percent is transferred into the reactor2) Sterilizing for 8min, repeatedly washing with sterile water, and drying with sterilized paper. Placing the sterilized leaves in a culture dish, cutting off the periphery of the leaves, vertically cutting 1 knife on the main vein of the leaves, and inoculating the leaves on a callus culture medium.
2. Induction of callus
The formula of the culture medium for inducing the callus tissue comprises the following components: MS + BA 0.5mg/L + IAA 2.0mg/L + banana 100g/L + sucrose 30g/L + agar 7g/L, pH 6.0. Each flask was inoculated with 5 explants and 20 flasks. The culture conditions were: culturing at 20 deg.C in dark place in sterile culture room. Inoculating the explants on a callus culture medium, culturing for 30 days, starting to germinate the callus, counting the number of the callus after 50 days, and recording the callus induction rate, wherein the callus induction rate is the number of explants inducing the callus/the number of inoculated explants. The result shows that the leaf of euonymus alatus is used as the explant on the callus culture medium to be tested, the callus induction rate reaches 98.5%, and the callus has good appearance, the size is about 5mm, the texture is compact, and the color is light yellow green.
3. Differentiation culture of callus
Cutting the excellent callus into small blocks with the diameter of 4mm, inoculating the small blocks into a differentiation culture medium for differentiation culture, wherein the culture conditions are as follows: the temperature is 20 ℃, the illumination intensity is 1500lx, the illumination time is 10 h/day, and the culture place is an aseptic culture room. The formula of the differentiation medium is as follows: MS + BA (2.0mg/L) + BR (0.5mg/L) + IAA (0.2mg/L) + banana puree (100g/L) + sucrose (30g/L) + agar (7g/L), pH 6.0. 5 callus pieces were inoculated per bottle, 20 bottles were inoculated. After the callus is cultured by a differentiation medium, a certain number of buds germinate on the callus, and after 25 days, the differentiated buds are observed and counted to calculate the differentiation rate (the number of differentiated buds/the number of inoculated materials). BR is brassinolide, a natural cytokinin. As a result, the differentiation rate of the euonymus alatus callus is 96.8 percent after the euonymus alatus callus is subjected to differentiation culture on a tested differentiation culture medium, the height of a differentiated bud is about 3mm, and the bud is bright green. After 3 times of differentiation subculture, the propagation coefficient is 6 times, and strong seedling culture is carried out.
4. Strong seedling culture
Because the buds cultured by differentiation are tender, certain strong seedling culture is necessary. Cutting off buds on a differentiation culture medium, and inoculating the buds on a strong seedling culture medium for strong seedling culture. The culture conditions were: the temperature is 23 ℃, the illumination intensity is 2000lx, the illumination time is 12 h/day, and the culture place is an aseptic culture room. The formula of the strong seedling culture medium is as follows: MS + BA (2.0mg/L) + BR (0.5mg/L) + IAA (0.1mg/L) + banana puree (100g/L) + sucrose (20g/L) + agar (7.0g/L), pH 5.8. 5 plantlets with consistent growth are inoculated in each bottle, and 20 plantlets are inoculated. The growth of the seedlings was observed after one month for 30 days. The result shows that the Euonymus alatus buds grow robustly and are bright green in color and luster and 6-8cm in height after being cultured by the strong seedling culture medium for testing.
5. Rooting culture
Cutting the strong euonymus alatus rootless seedlings into two sections, and transferring the two sections to a rooting culture medium for culture. The culture conditions were: the temperature is 27 ℃, the illumination intensity is 2500lx, the illumination time is 15 h/day, and the culture place is an aseptic culture room. The rooting medium comprises the following components: 1/2MS + BR (0.1mg/L) + IAA (1.0mg/L) + sucrose 30g/L + agar 7g/L, pH 6.0. Each bottle was inoculated with 5 seedling stems (grown consistently and about 4cm in height) and 20 bottles were inoculated. The result shows that after the euonymus alatus tissue culture seedlings are cultured on the rooting culture medium for the test, white roots can be generated after 2 weeks, the rooting rate reaches 100%, the roots grow strongly after 30 days, the length reaches 5-7cm, and 5-7 adventitious roots are differentiated from each seedling. Hardening and transplanting the seedlings after 60 days.
6. Hardening and transplanting of euonymus alatus
The euonymus alatus tissue culture seedling exercising can be carried out by unscrewing the bottle cap in a greenhouse at the temperature of 22 ℃ and placing for 10 days, and then carrying out warm-bed transition transplanting. The transition seedbed (namely hotbed) is built in a common single plastic greenhouse, and the culture medium is sandy soil: peat soil: the vermiculite is used after being fully and uniformly mixed according to the volume ratio of 3:4:2, and the culture medium is used after being sterilized and fully and uniformly mixed. When transplanting, the seedlings are taken out from the bottle, the solid culture medium at the roots of the euonymus alatus tissue culture seedlings is cleaned by clear water, and meanwhile, the root damage is reduced as much as possible. After the seeds are planted in the seedbed, clean water is selected for irrigation, 1 time of pesticide liquid diluted 1000 times by mass of 75% chlorothalonil wettable powder is sprayed every two weeks for disinfection, and 4 times of continuous spraying is carried out. In the early stage of transplanting, special attention is paid to keeping the seedbed and the air humidity, the humidity is kept at 83 percent, the temperature is 25 ℃, and shading is carried out for 7 days in the early stage; the whole is managed for 8 days, and the ventilation is gradually carried out for 20 days according to the condition, and the covering is removed. The survival rate reaches 97.5 percent.
Example 2
1. Sterilizing and disinfecting
Selecting a strong seedling plant which grows for three years, and selecting the 4 th leaf of the plant from the top of the plant as an explant.
Repeatedly washing explant under running water for 40min, adding appropriate amount of detergent, soaking while shaking for 40min, washing, and transferring to ultra-clean stateThe working platform is used for carrying out the next disinfection treatment. The disinfection treatment comprises the following steps: soaking the explant in 80% alcohol for 5s, and repeatedly washing the explant with sterile water for 4 times, wherein the washing time is 10min each time; then transferring the explant into NaClO solution with the mass concentration of 12% for disinfection for 10min, and repeatedly washing and oscillating the explant for 4 times by using sterile water, wherein the washing time is 10 each time; finally, mercury mercuric chloride (HgCl) with the mass concentration of 0.1 percent is transferred into the reactor2) Sterilizing for 5min, repeatedly washing with sterile water, and drying with sterilized paper. Placing the sterilized leaves in a culture dish, cutting off the periphery of the leaves, vertically cutting 2 knives on the main veins of the leaves, and inoculating the leaves on a callus culture medium.
2. Induction of callus
The formula of the culture medium for inducing the callus tissue comprises the following components: MS + BA (0.2-0.5mg/L) + IAA (1.0mg/L) + banana puree (50g/L) + sucrose (20g/L) + agar (6.5g/L), pH 5.8. Each flask was inoculated with 5 explants and 20 flasks. The culture conditions were: the temperature is 24 ℃, the culture is carried out in dark, and the culture place is an aseptic culture room. Inoculating the explants on a callus culture medium for 35 days, counting the number of the callus after 55 days of callus germination, and recording the callus induction rate, wherein the callus induction rate is the number of the explants inducing the callus/the number of the inoculated explants. The result shows that the leaf of euonymus alatus is used as the explant on the callus culture medium to be tested, the callus induction rate reaches 97.8 percent, and the callus has good appearance, the size is about 7mm, the texture is compact, and the color is light yellow green.
3. Differentiation culture of callus
Cutting the excellent callus into small pieces of 2mm, inoculating into a differentiation culture medium for differentiation culture, wherein the culture conditions are as follows: the temperature is 24 ℃, the illumination intensity is 2000lx, the illumination time is 12 h/day, and the culture place is an aseptic culture room. The formula of the differentiation medium is as follows: MS + BA (1.0mg/L) + BR (0.1mg/L) + IAA (0.1mg/L) + banana puree (50g/L) + sucrose (20g/L) + agar (6.5g/L), pH 5.8. 5 callus pieces were inoculated per bottle, 20 bottles were inoculated. After the callus is cultured by a differentiation medium, a certain number of buds germinate on the callus, and after one month of 25-30 days, the differentiated buds are observed and counted to calculate the differentiation rate (the number of differentiated buds/the number of inoculated materials). BR is brassinolide, a natural cytokinin. As a result, the differentiation rate of the euonymus alatus callus after the differentiation culture on the tested differentiation medium is 95.0%, the height of the differentiated bud is about 4mm, and the bud is bright green. After differentiation subculture, the propagation coefficient is 7 times, and strong seedling culture is carried out.
4. Strong seedling culture
Because the buds cultured by differentiation are tender, certain strong seedling culture is necessary. Cutting off buds on a differentiation culture medium, and inoculating the buds on a strong seedling culture medium for strong seedling culture. The culture conditions were: the temperature is 27 ℃, the illumination intensity is 2500lx, the illumination time is 15 h/day, and the culture place is an aseptic culture room. The formula of the strong seedling culture medium is as follows: MS + BA (3.0mg/L) + BR (1.0mg/L) + IAA (0.2mg/L) + banana puree (150g/L) + sucrose (30g/L) + agar (7.5g/L), pH 6.0. 5 plantlets with consistent growth are inoculated in each bottle, and 20 plantlets are inoculated. The growth of the seedlings was observed after 35 days and one month. The result shows that the Euonymus alatus buds grow robustly and are bright green in color and luster and 6-8cm in height after being cultured by the strong seedling culture medium for testing.
5. Rooting culture
Cutting the strong euonymus alatus rootless seedlings into two sections, and transferring the two sections to a rooting culture medium for culture. The culture conditions were: the temperature is 23 ℃, the illumination intensity is 2000lx, the illumination time is 12 h/day, and the culture place is an aseptic culture room. The rooting medium comprises the following components: 1/2MS + BR (0.05mg/L) + IAA (0.5mg/L) + sucrose (28g/L) + agar (8g/L), pH 5.8. Each bottle was inoculated with 5 seedling stems (grown consistently and about 3cm in height) and 20 bottles were inoculated. The result shows that after the tissue culture seedlings of euonymus alatus are cultured on the rooting culture medium for testing, white roots can be generated after 2 weeks, the rooting rate reaches 100%, the roots grow strongly after 30 days, the length reaches 5-7cm, and 5-7 adventitious roots can be differentiated from each seedling. Hardening and transplanting the seedlings after 50 days.
6. Hardening and transplanting of euonymus alatus
The euonymus alatus tissue culture seedling exercising can be carried out by unscrewing the bottle cap in a greenhouse at the temperature of 18 ℃ and placing for 7 days, and then carrying out warm-bed transition transplanting. The transition seedbed (namely hotbed) is built in a common single plastic greenhouse, and the culture medium is sandy soil: peat soil: the vermiculite is used after being fully and uniformly mixed according to the volume ratio of 4:4:1, and the culture medium is used after being sterilized and fully and uniformly mixed. When transplanting, the seedlings are taken out from the bottle, the solid culture medium at the roots of the euonymus alatus tissue culture seedlings is cleaned by clear water, and meanwhile, the root damage is reduced as much as possible. After the seeds are planted in the seedbed, clean water is selected for irrigation, 1 time of pesticide liquid diluted by 1500 times of chlorothalonil wettable powder with the mass percent of 75% is sprayed every two weeks for disinfection, and 3 times of continuous spraying is carried out. In the early stage of transplanting, special attention is paid to keeping the seedbed and the air humidity, the humidity is kept at 87%, the temperature is 21 ℃, and shading is carried out for 10 days in the early stage; the whole is generally managed for 12 days or so, and the ventilation is gradually carried out for 20 days according to the condition, and the covering is removed. The survival rate can reach 98.1 percent.
Example 3
1. Sterilizing and disinfecting
Selecting robust seedling plants which grow for three years, and selecting the 3 rd leaf of the plant from the top of the plant as an explant.
Repeatedly washing the explant under running water for 35min, adding a proper amount of detergent, soaking for 35min while oscillating, washing, and transferring to a clean bench for the next disinfection treatment. The disinfection treatment comprises the following steps: soaking the explant in 78% alcohol for 7s, and repeatedly washing the explant with sterile water for 5 times, wherein the washing time is 8min each time; then transferring the explant into NaClO solution with mass concentration of 11% for disinfection for 13min, and repeatedly washing and oscillating the explant for 5 times by using sterile water, wherein the washing time is 8min each time; finally, mercury mercuric chloride (HgCl) with the mass concentration of 0.15 percent is transferred into the reactor2) Sterilizing for 7min, repeatedly washing with sterile water, and drying with sterilized paper. Placing the sterilized leaves in a culture dish, cutting off the periphery of the leaves, vertically cutting 2 knives on the main veins of the leaves, and inoculating the leaves on a callus culture medium.
2. Induction of callus
The formula of the culture medium for inducing the callus tissue comprises the following components: MS + BA (0.4mg/L) + IAA (1.5mg/L) + banana puree (75g/L) + sucrose (25g/L) + agar (6.7g/L), pH 5.9. Each flask was inoculated with 5 explants and 20 flasks. The culture conditions were: the temperature is 22 ℃, the culture is carried out in dark, and the culture place is an aseptic culture room. Inoculating the explants on a callus culture medium, culturing for 33 days, starting to germinate the callus, counting the number of the callus after 53 days, and recording the callus induction rate, wherein the callus induction rate is the number of explants inducing the callus/the number of inoculated explants. As a result, it was found that when the leaves of Euonymus alatus were used as explants in the callus culture medium to be tested, the callus induction rate reached 96.9%, and the callus exhibited good appearance, about 7mm in size, dense texture, and pale yellow-green color. BR is brassinolide, a natural cytokinin.
3. Differentiation culture of callus
Cutting the excellent callus into small pieces with the diameter of 3mm, inoculating the small pieces into a differentiation culture medium for differentiation culture, wherein the culture conditions are as follows: the temperature is 22 ℃, the illumination intensity is 1700lx, the illumination time is 11 h/day, and the culture place is an aseptic culture room. The formula of the differentiation medium is as follows: MS + BA (1.5mg/L) + BR (0.4mg/L) + IAA (0.15mg/L) + Banana puree (75g/L) + sucrose (25g/L) + agar (6.7g/L), pH 5.9. 5 callus pieces were inoculated per bottle, 20 bottles were inoculated. After the callus is cultured by the differentiation medium, a certain number of buds germinate on the callus, and the differentiation rate (the number of differentiated buds/the number of inoculated materials) is calculated by observing and counting the differentiated buds after 28 days. As a result, the differentiation rate of the euonymus alatus callus after the differentiation culture on the tested differentiation medium is 98.1%, the height of the differentiated bud is about 3mm, and the bud is bright green. After 4 differentiation subculture, the propagation coefficient is 7 times, and strong seedling culture is carried out.
4. Strong seedling culture
Because the buds cultured by differentiation are tender, certain strong seedling culture is necessary. Cutting off buds on a differentiation culture medium, and inoculating the buds on a strong seedling culture medium for strong seedling culture. The culture conditions were: the temperature is 25 ℃, the illumination intensity is 2200 lx, the illumination time is 13 h/day, and the culture place is an aseptic culture room. The formula of the strong seedling culture medium is as follows: MS + BA (2.5mg/L) + BR (0.8mg/L) + IAA (0.15mg/L) + banana puree (125g/L) + sucrose (25g/L) + agar (7.25g/L), pH 5.9. 5 plantlets with consistent growth are inoculated in each bottle, and 20 plantlets are inoculated. After 35 days, the growth of the seedlings was observed. The result shows that the Euonymus alatus buds grow robustly and are bright green in color and luster and 6-8cm in height after being cultured by the strong seedling culture medium for testing.
5. Rooting culture
Cutting the strong euonymus alatus rootless seedlings into two sections, and transferring the two sections to a rooting culture medium for culture. The culture conditions were: the temperature is 25 ℃, the illumination intensity is 2300lx, the illumination time is 13 h/day, and the culture place is an aseptic culture room. The rooting medium comprises the following components: 1/2MS + BR (0.07mg/L) + IAA (0.8mg/L) + sucrose (29g/L) + agar (7.5g/L), pH 5.9. Each bottle was inoculated with 5 seedling stems (grown consistently and about 3-4cm in height) and 20 bottles were inoculated. The result shows that after the tissue culture seedlings of euonymus alatus are cultured on the rooting culture medium for testing, white roots can be generated after 2 weeks, the rooting rate reaches 100%, the roots grow strongly after 30 days, the length reaches 5-7cm, and 5-7 adventitious roots can be differentiated from each seedling. Hardening and transplanting after 60 days.
6. Hardening and transplanting of euonymus alatus
The euonymus alatus tissue culture seedling exercising can be carried out by unscrewing the bottle cap in a greenhouse at the temperature of 20 ℃ and placing for 8 days, and then carrying out warm-bed transition transplanting. The transition seedbed (namely hotbed) is built in a common single plastic greenhouse, and the culture medium is sandy soil: peat soil: the vermiculite is used after being fully and uniformly mixed according to the volume ratio of 3:4:1, and the culture medium is used after being sterilized and fully and uniformly mixed. When transplanting, the seedlings are taken out from the bottle, the solid culture medium at the roots of the euonymus alatus tissue culture seedlings is cleaned by clear water, and meanwhile, the root damage is reduced as much as possible. After the seeds are planted in the seedbed, clean water is selected for irrigation, 1 time of pesticide liquid diluted by 1200 times of chlorothalonil wettable powder with the mass percent of 80 percent is sprayed every two weeks for disinfection, and 3 times of continuous spraying is carried out. In the early stage of transplanting, special attention is paid to keeping the seedbed and the air humidity, the humidity is kept at 85 percent, the temperature is 23 ℃, and shading is carried out for 9 days in the early stage; the whole is managed for 10 days, and the ventilation is gradually carried out for 20 days according to the condition, and the covering is removed. The survival rate reaches 98.1 percent.
Claims (9)
1. A culture method of euonymus alatus seedlings is characterized by comprising the following steps:
(1) explant disinfection: selecting leaves at the top of a euonymus alatus plant, and sterilizing the explant by sequentially using alcohol, NaClO solution and mercury bichloride to obtain a sterilized explant;
(2) induction of callus: inoculating the sterilized explants obtained in the step (1) on a callus culture medium for culture to obtain callus; the callus culture medium comprises the following components in percentage by weight: MS, 0.2-0.5mg/L BA, 1.0-2.0 mg/LIAA, 50-100 g/L banana puree, 20-30g/L sucrose and 6.5-7g/L agar, wherein the pH value is 5.8-6.0, the culture temperature is 20-24 ℃, and the culture time is 50-55 days;
(3) differentiation culture of callus: cutting the callus in the step (2) into small pieces, and inoculating the small pieces into differential culture to perform differential culture to obtain euonymus alatus buds;
(4) strong seedling culture: cutting the euonymus alatus buds obtained in the step (3) from a differentiation culture medium, inoculating the euonymus alatus buds on a strong seedling culture medium, and performing strong seedling culture to obtain euonymus alatus rootless seedlings;
(5) rooting culture: segmenting the euonymus alatus rootless seedlings obtained in the step (4), and transferring the euonymus alatus rootless seedlings to a rooting culture medium for culture to obtain seedlings with adventitious roots;
(6) hardening and transplanting seedlings: and (5) planting the seedlings with the adventitious roots obtained in the step (5) into a transition seedbed, irrigating and disinfecting, and culturing euonymus alatus seedlings.
2. The method of claim 1, wherein: soaking the obtained product in 75-80 vol% alcohol for 5-10 s, and washing with water; the NaClO solution is sterilized by using a NaClO solution with the mass concentration of 10-12% for 10-15 min and then washed by water; the mercuric chloride disinfection is carried out for 5-8 min by using mercuric chloride with the mass concentration of 0.1-0.2%.
3. The method of claim 1, wherein: the culture medium for the differentiation culture in the step (3) is MS, 1.0-2.0 mg/L BA, 0.1-0.5 mg/L BR, 0.1-0.2mg/L IAA, 50-100 g/L banana puree, 20-30g/L sucrose and 6.5-7g/L agar, the pH value is 5.8-6.0, the culture temperature is 20-24 ℃, the culture time is 25-30 days, and the culture times are 3-4 times of differentiation subculture; the differential culture is illumination culture, the illumination intensity of the illumination culture is 1500-2000 lx, and the illumination time is 10-12 h every day.
4. The method of claim 1, wherein: the culture medium for strong seedling culture in the step (4) comprises MS, 2.0-3.0mg/L BA, 0.5-1.0mg/L BR, 0.1-0.2mg/L IAA, 100-150g/L banana puree, 20-30g/L sucrose and 7.0-7.5g/L agar, the pH value is 5.8-6.0, the culture temperature is 23-27 ℃, and the culture time is 30-35 days; the culture is aseptic culture under the illumination condition, the illumination intensity of the illumination culture is 2000-2500 lx, and the illumination time is 12-15 h every day.
5. The method of claim 1, wherein: the rooting culture medium in the step (5) is 1/2MS, 0.05-0.1 mg/L BR, 0.5-1.0mg/L IAA, 28-30 g/L sucrose and 7-8 g/L agar, the pH value is 5.8-6.0, the culture temperature is 23-27 ℃, and the culture time is 50-60 days; the culture is aseptic culture under the illumination condition, the illumination intensity of the illumination culture is 2000-2500 lx, and the illumination time is 12-15 h every day.
6. The method of claim 1, wherein: placing the seeds in the step (6) at the temperature of 18-22 ℃ for 7-10 days before entering the transition seedbed.
7. The method of claim 1, wherein: the cultivation medium of the transition seedbed in the step (6) is a mixture of sandy soil, peat soil and vermiculite, the volume ratio of which is (3-4) to (2-4) to (1-2) in sequence, the cultivation humidity in the transition seedbed is 83-87%, and the cultivation temperature is 21-25 ℃.
8. The method of claim 1, wherein: the disinfection in the step (6) is implemented by using 1000-1500 times of liquid medicine diluted by 75-80% of chlorothalonil wettable powder, and continuously spraying for 3-4 times; the disinfection frequency is once every 14 to 17 days.
9. The method of claim 1, wherein: shading 7-10 days after the cultivation in the transition seedbed, and ventilating and removing the covering after the cultivation in the closed space for 8-12 days.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010342971.XA CN111670807B (en) | 2020-04-27 | 2020-04-27 | Tissue culture method of euonymus alatus |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010342971.XA CN111670807B (en) | 2020-04-27 | 2020-04-27 | Tissue culture method of euonymus alatus |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN111670807A CN111670807A (en) | 2020-09-18 |
| CN111670807B true CN111670807B (en) | 2022-03-11 |
Family
ID=72452262
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010342971.XA Active CN111670807B (en) | 2020-04-27 | 2020-04-27 | Tissue culture method of euonymus alatus |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111670807B (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105850743A (en) * | 2016-05-04 | 2016-08-17 | 沈阳农业大学 | Winged euonymus stem adventitious bud inducing method |
| CN106258973A (en) * | 2016-08-17 | 2017-01-04 | 北京林业大学 | A kind of red autumnal leaves winged euonymus polyploid cell breeding technique |
| CN106489738A (en) * | 2016-11-07 | 2017-03-15 | 金陵科技学院 | A kind of production method of spindle tree leaf regeneration plant |
| CN106577283A (en) * | 2016-12-14 | 2017-04-26 | 河南红枫种苗股份有限公司 | Medium and method for tissue culture of euonymus maackii |
| CN107306795A (en) * | 2017-08-08 | 2017-11-03 | 佛山科学技术学院 | A kind of efficient Euonymus fortunei regeneration culture medium group |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20130205449A1 (en) * | 2011-08-04 | 2013-08-08 | University Of Connecticut | Methods and Compositions for Production of Triploid Sterile Plants |
| CN111406524A (en) * | 2020-04-26 | 2020-07-14 | 江苏农林职业技术学院 | Cutting propagation method of euonymus alatus |
-
2020
- 2020-04-27 CN CN202010342971.XA patent/CN111670807B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105850743A (en) * | 2016-05-04 | 2016-08-17 | 沈阳农业大学 | Winged euonymus stem adventitious bud inducing method |
| CN106258973A (en) * | 2016-08-17 | 2017-01-04 | 北京林业大学 | A kind of red autumnal leaves winged euonymus polyploid cell breeding technique |
| CN106489738A (en) * | 2016-11-07 | 2017-03-15 | 金陵科技学院 | A kind of production method of spindle tree leaf regeneration plant |
| CN106577283A (en) * | 2016-12-14 | 2017-04-26 | 河南红枫种苗股份有限公司 | Medium and method for tissue culture of euonymus maackii |
| CN107306795A (en) * | 2017-08-08 | 2017-11-03 | 佛山科学技术学院 | A kind of efficient Euonymus fortunei regeneration culture medium group |
Non-Patent Citations (4)
| Title |
|---|
| "扶芳藤的组织培养和快速繁殖的研究";尹淑萍等;《全国植物组培、脱毒快繁及工厂化生产技术学术研讨会论文集》;20010901;第221页第1.2-1.3节，第222页第2.1-2.4节， * |
| "扶芳藤组织培养再生体系建立研究";徐萌;《中国优秀硕士学位论文全文数据库 (电子期刊) 农业科技辑》;20050815(第04期);第9页第2.2.1节 * |
| "金叶卫矛无菌系建立及继代培养基配方优化";王洪波等;《山东林业科技》;20081215(第6期);第46页表1 * |
| "金叶卫矛的茎段培养及试管繁殖";于萍等;《园艺学报》;20000225;第27卷(第1期);第69页第1节第2-3段，第2.2-2.4节 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN111670807A (en) | 2020-09-18 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108293878B (en) | Tissue culture seedling raising method for trichosanthes kirilowii Maxim tender leaves | |
| CN105165368A (en) | Method for rapidly breeding seedlings by using bletilla striata tubers | |
| CN111616052A (en) | Rapid propagation and sugar-free rooting culture method and application of apple rootstock catalpa bungei | |
| CN110192524A (en) | A method of using base of leaf and peduncle kryptoblast as the in vitro fast breeding of the zingiberaceous plant of explant | |
| CN108419675B (en) | Tissue culture seedling raising method for passion fruit top shoots | |
| CN114902961A (en) | Method for creating new ornamental pineapple variety by adopting EMS mutagenesis | |
| CN104855292A (en) | Method for tissue culture and rapid propagation of stems of cinnamomum kanehirae hay | |
| CN106359087A (en) | Tissue culture quick-breeding seedling raising method for radix asparagi | |
| CN112655553A (en) | Rapid sterile short-shoot propagation method for Orthosiphon aristatus | |
| CN107660464A (en) | A kind of tissue culture and rapid propagation method of sequoia sempervirens good seed | |
| CN112243861A (en) | Tissue culture and rapid propagation method for Huagaimu | |
| CN108271693B (en) | Method for quickly establishing and storing regeneration system of parasitic orchid | |
| CN109156350B (en) | Anti-aleurites fordii propagation bud and rooting culture medium and method for promoting in-vitro rapid propagation of anti-aleurites fordii | |
| CN108391591B (en) | Tissue culture and rapid propagation method for tabebuia flavedo | |
| CN108651284B (en) | Method for inducing fast callus of machilus thunbergii | |
| CN114946655B (en) | Liupao tea seedling tissue culture method | |
| CN111670807B (en) | Tissue culture method of euonymus alatus | |
| CN110999782A (en) | Tissue culture and rapid propagation method for gardenia jasminoides | |
| CN112243860B (en) | Tissue culture and rapid propagation method for Chinese parasol trees | |
| CN102884980B (en) | Quick tissue propagation method of paphiopedilum bellatulum | |
| CN111480577B (en) | Tissue culture rapid propagation method for epimedium wushanense inflorescence | |
| CN112352678B (en) | Tissue culture rapid propagation technology for slash pine seedlings | |
| CN103621400A (en) | Toona sinensis bud soilless cultivation method | |
| CN1541518A (en) | Dendrobium unicum aseptic seeding and test tube seedling tecnnology | |
| CN113287518A (en) | Commercial hippeastrum rutilum seed ball production method in south China |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |