CN113100060A - Tissue culture propagation method for alpine rhododendron - Google Patents

Tissue culture propagation method for alpine rhododendron Download PDF

Info

Publication number
CN113100060A
CN113100060A CN202110425793.1A CN202110425793A CN113100060A CN 113100060 A CN113100060 A CN 113100060A CN 202110425793 A CN202110425793 A CN 202110425793A CN 113100060 A CN113100060 A CN 113100060A
Authority
CN
China
Prior art keywords
rooting
bud
culture
adventitious
rhododendron
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.)
Pending
Application number
CN202110425793.1A
Other languages
Chinese (zh)
Inventor
李玲莉
邹世慧
汤丽红
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
CHONGQING LANDSCAPE AND GARDENING RESEARCH INSTITUTE
Original Assignee
CHONGQING LANDSCAPE AND GARDENING RESEARCH INSTITUTE
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by CHONGQING LANDSCAPE AND GARDENING RESEARCH INSTITUTE filed Critical CHONGQING LANDSCAPE AND GARDENING RESEARCH INSTITUTE
Priority to CN202110425793.1A priority Critical patent/CN113100060A/en
Publication of CN113100060A publication Critical patent/CN113100060A/en
Pending legal-status Critical Current

Links

Classifications

    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01HNEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H4/00Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
    • A01H4/008Methods for regeneration to complete plants
    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01HNEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H4/00Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
    • A01H4/001Culture apparatus for tissue culture

Abstract

The invention discloses a tissue culture propagation method of alpine rhododendron, which comprises the following steps: s1, explant induction: inoculating a sterile explant of rhododendron lapponicum into an induction culture medium, and inducing to generate an adventitious bud cluster; s2, proliferation and strong seedling culture: cutting the obtained adventitious bud cluster, inoculating into a strong seedling culture medium, and culturing into adventitious buds; s3, rooting culture: inoculating the adventitious bud after the strong seedling culture into a rooting culture medium, and culturing for 28-30 days; then taking out the adventitious bud and inoculating the adventitious bud into a loose porous rooting matrix, culturing under an aseptic condition, inducing to generate adventitious roots after 10-15 days, and growing into a rooting seedling with a complete root system after 50-60 days; s4, transplanting rooting seedlings: transplanting the rooted seedlings to obtain survival rhododendron lapponicum tissue culture seedlings; the method provided by the application carries out rooting culture on the adventitious buds cultured by strong seedling, and cultures the adventitious buds into rooting seedlings with complete root systems, so that the transplanting success rate of alpine rhododendron is improved.

Description

Tissue culture propagation method for alpine rhododendron
Field of the method
The invention belongs to the field of plant tissue culture methods, and particularly relates to a tissue culture propagation method of alpine rhododendron.
Background method
The alpine rhododendron is originally produced in China, in recent years, with the rapid development of the domestic flower market, potted flowers of alpine rhododendron not only appear on the important occasions where national leaders meet foreign guests for many times, but also serve as high-grade night flowers, are pursued by consumers, and the selling price of each pot is 300-1000 yuan, even up to 8800 yuan.
At present, potted rhododendron at China mainly depends on import from Germany and Belgium, and the price is high; in the current original producing areas of alpine rhododendron in China, only a few enterprises develop seedling production and potted flower production. In foreign countries, rhododendron lapponicum seedling production enterprises mainly carry out large-scale production in a tissue culture mode; in China, the rhododendron lapponicum seedlings are mainly produced by sowing and raising seedlings, because the seeds of the rhododendron lapponicum are extremely small, the rhododendron lapponicum can germinate by means of moss, and the seedlings grow slowly, the large-scale production of specific species or varieties of the rhododendron lapponicum is limited.
When the existing alpine rhododendron tissue culture is carried out, a stem section of the alpine rhododendron is taken as an explant, the explant is placed in ethanol for soaking and disinfection, and after the explant is washed by sterile water for many times and is sterilized by a sterilizing agent again, the sterilization of the explant is completed by continuously washing by the sterile water; then placing the explant after soaking and sterilization in an induction culture medium to induce and generate adventitious buds, finally washing the culture medium from the adventitious buds generated by induction, and then exercising and rooting outside a bottle. In the culture method, on one hand, the disinfection mode has complex steps and long disinfection time, and the rhododendron lapponicum has more body hair and the body hair is distributed at stem sections and the like, so that the traditional disinfection and sterilization process is difficult to achieve thorough disinfection and causes great damage to explants; on the other hand, the adventitious bud generated by induction is directly cultured outside the bottle to root, and the rooting culture mode needs to be carried out in a harsher temperature and humidity environment, and has longer rooting time and lower rooting rate.
The Chinese invention patent with the publication number of CN102119660A discloses a method for rooting culture and hardening seedling transplantation in a greenhouse of rhododendron lapponicum tissue culture seedlings, which is characterized in that after bottle seedlings grow out, the seedlings are transplanted after rooting culture in the greenhouse by adopting a water culture rooting method, and the rooting rate and the transplanting survival rate of the rhododendron lapponicum tissue culture seedlings are improved to a certain extent.
Disclosure of Invention
In order to solve the problems of the existing methods, the invention aims to provide a rhododendron lapponicum tissue culture propagation method.
The method scheme adopted by the invention is as follows:
a tissue culture propagation method of rhododendron lapponicum comprises the following steps:
s1, explant induction: inoculating a sterile explant of rhododendron lapponicum into an induction culture medium, and inducing to generate an adventitious bud cluster;
s2, proliferation and strong seedling culture: cutting the generated adventitious bud cluster, inoculating the cut adventitious bud cluster into a strong seedling culture medium, and culturing the adventitious bud cluster into adventitious buds;
s3, rooting culture: inoculating the adventitious bud after the strong seedling culture into a rooting culture medium, and culturing for 28-30 days; then taking out the adventitious bud and inoculating the adventitious bud into a loose porous rooting matrix, culturing under an aseptic condition, inducing to generate adventitious roots after 10-15 days, and growing into a rooting seedling with a complete root system after 50-60 days;
s4, transplanting rooting seedlings: transplanting the rooted seedlings to obtain survival rhododendron lapponicum tissue culture seedlings.
Preferably, the sterile explant is the bud or/and terminal bud of rhododendron lapponicum.
Preferably, in step S1, the sterile explant is obtained by the following method:
selecting a flower bud which has a length of 4-5 cm, is not expanded and blooms and is kept in a perfect aseptic environment inside, spraying an ethanol solution on the surface of the flower bud for sterilization, and circularly peeling bracts of the flower bud layer by using an aseptic blade in the aseptic environment to obtain an aseptic flower bud or/and an aseptic terminal bud, wherein the aseptic flower bud or/and the aseptic terminal bud are aseptic explants.
Preferably, in the step S1, the non-contaminated explant is cut 10-14 days after the sterile explant is inoculated into the induction medium, and then is inoculated into the induction medium again, and 80-90 days later, the callus is obtained and adventitious bud clumps are formed.
Preferably, in the step S4, when the rooted seedlings are transplanted, the rooted seedlings with complete root systems are transplanted into a seedling hardening transplanting substrate which is obtained by mixing turf and perlite and has a pH value of 5.9-7.0, and the tissue culture seedlings of the survived alpine rhododendron are obtained after culturing for 30-40 days.
Preferably, in the S1, the induction medium consists of WPM medium + TDZ + NAA + agar + sucrose;
the mass concentration of the TDZ is 0.5-2 mg.L-1(ii) a The mass concentration of NAA is 0-0.5 mg.L-1(ii) a The mass concentration of agar is 7 g.L-1(ii) a The mass concentration of sucrose is 30 g.L-1
The pH of the induction medium was 5.8.
Preferably, in S2, the culture medium for proliferation and strong seedling is WPM culture medium + TDZ + IBA + GA3+ agar + sucrose;
the mass concentration of the TDZ is 0-0.1 mg.L-1(ii) a The mass concentration of IBA is 0-0.1 mg.L-1;GA3Has a mass concentration of 5 to 7 mg.L-1(ii) a The mass concentration of agar is 7 g.L-1(ii) a The mass concentration of sucrose is 30 g.L-1
The pH value of the proliferation and strong seedling culture medium is 5.8.
Preferably, in the S3, the rooting medium is composed of WPM medium + IBA + agar + sucrose,
the mass concentration of IBA is 0.5-1 mg.L-1(ii) a The mass concentration of agar is 7 g.L-1(ii) a The mass concentration of sucrose is 30 g.L-1
The pH value of the rooting medium is 5.8.
Preferably, in S3, the loose porous rooting matrix is loose porous perlite and/or loose porous vermiculite.
Preferably, in S4, when the rooted seedlings are transplanted, the rooted seedlings are removed from the sterile environment and cultured in a cool and ventilated place with the relative humidity of air not lower than 80%.
The invention has the beneficial effects that:
according to the method, after the alpine rhododendron explant is induced, proliferated and cultured into a thick and strong adventitious bud through strong seedling culture, the adventitious bud is inoculated into a rooting culture medium for rooting culture, and the adventitious bud is cultured for 28-30 days to fully absorb nutrition. Meanwhile, the adventitious bud can grow further in the rooting culture medium, and a nutrient bank is synchronously constructed in the body of the adventitious bud to store nutrient substances; then taking out the adventitious bud, placing the adventitious bud in a loose porous rooting matrix, culturing under an aseptic condition, culturing a complete root system with an active nutrition absorption function under the aseptic condition by utilizing the improved rooting matrix with air permeability and combining stock nutrition in the adventitious bud, improving the growth potential of the tissue culture seedling, enhancing the resistance of the tissue culture seedling to environmental stress in the seedling exercising and transplanting process, improving the transplanting survival rate, and solving the problems of slow rooting outside a tissue culture seedling bottle, long seedling exercising and transplanting time, low survival rate and the like in the large-scale production of the specific variety of rhododendron lapponicum.
In the tissue culture propagation process, the culture material is always in an aseptic environment, and the tissue culture seedling can not move out of the aseptic environment until the tissue culture seedling is acclimatized and transplanted. The whole tissue culture steps are few, the flow is simple, the addition of various additives and the like is effectively reduced, the self nutrition is fully utilized to achieve the rooting effect, the cost is saved, the energy consumption is reduced, and the tissue culture method has the characteristics of environmental friendliness, strong practicability and applicability and the like.
Drawings
FIG. 1 is a schematic diagram showing adventitious bud induction in the example of tissue culture of Rhododendron lapponicum;
FIG. 2 is a schematic diagram of strong seedling culture in the Rhododendron lapponicum tissue culture example;
FIG. 3 is a schematic view of rooting culture of the Rhododendron lapponicum tissue culture example;
FIG. 4 is a schematic view of the tissue culture example of Rhododendron lapponicum seedlings with intact root systems;
FIG. 5 is a schematic diagram of an example of the tissue culture seedling of alpine rhododendron which survives after the transplantation of the culture seedling.
Detailed Description
The present invention is further illustrated below with reference to specific examples. Those skilled in the art will appreciate that the following described embodiments are illustrative of some, but not all, embodiments of the invention and are not to be construed as limiting the scope of the invention. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention. The examples were carried out under the conventional conditions, unless otherwise specified. The reagents used are all conventional products which are commercially available.
The tissue culture propagation method of rhododendron lapponicum comprises the following steps:
s1, explant induction: inoculating the aseptic explant of rhododendron lapponicum into an induction culture medium, and inducing to generate an adventitious bud cluster. The adventitious bud clumps induced are shown in FIG. 1.
The sterile explant can be obtained by adopting any tissue of alpine rhododendron which can induce to generate adventitious buds through disinfection and sterilization by a conventional method, and the explant is only required to be in a sterile state.
In one embodiment, the sterile explant is the bud of Rhododendron lapponicum and/or the terminal bud of the flower bud, which can be directly stripped from the flower bud in a sterile environment.
Specifically, in the period of 10-2 noon more than 5 days on a continuous sunny day, flower buds with flower branches are selected, and the buds are taken back to a laboratory after redundant leaves are cut off. Wherein, the flower branches are 4-5 cm long and are convenient to hold; the bud is required to grow strongly, expand and not bloom, and the interior of the bud is kept in a good sterile environment, namely the bud and the terminal bud in the bud are in the sterile environment; the diameter of the flower bud is about 1-3 cm. If the transportation time is too long, the explants should be well moisturized.
After the redundant petioles are broken off, 75-80% of ethanol solution is used for spraying wet surfaces of the buds and the flower branches, and the surfaces of the buds and the flower branches are sterilized to prevent bacteria or fungi on the surfaces of the buds and the flower branches from being brought into the explants when the explants are taken; then placing the sterilized flower buds in an aseptic environment, and peeling bracts layer by using an aseptic blade until the flower buds are peeled; and (3) stripping buds or/and terminal buds in the buds and inoculating the buds and/or the terminal buds in an induction culture medium.
Because buds and terminal buds in the flower buds are always wrapped by the buds and kept in an aseptic environment, and the buds are thallus-free, the application only needs to adopt a spraying mode on the outer surfaces of the flower buds, sterilize the surfaces of the flower buds by using an ethanol solution, and peel the wrapped buds layer by layer in the aseptic environment by using the aseptic environment in the flower buds, so that the aseptic buds and the terminal buds in the flower buds can be obtained and used as explants for tissue culture of rhododendron lapponicum for adventitious bud induction; the method has the advantages that the complex sterilization and disinfection treatment of the explants is not needed, the natural sterile explants in the flower buds are directly obtained, the operation is convenient and simple, the obtaining efficiency of the sterile explants is high, and the tissue culture efficiency of alpine rhododendron is improved to a certain extent.
After the sterile explant is stripped, inoculating the sterile explant into an induction culture medium, carrying out induction culture for 10-14 days, and cutting the cultured sterile explant to increase the induction area of the callus after determining that the sterile explant in the induction culture medium is in an uncontaminated state; then the cut explant is inoculated into an induction culture medium again, after the induction culture is continued for 80-90 days, the terminal bud, the petal, the filament, the anther, the flower stalk and the stigma of the bud can be induced to generate callus, and an adventitious bud cluster is formed. The differentiation time of the adventitious bud is different according to the species or variety of rhododendron lapponicum.
Wherein, the composition of the induction culture medium is as follows: WPM medium + TDZ 0.5-2 mg. L-1+NAA 0~0.5mg·L-1+ agar 7 g. L-1+ sucrose 30 g.L-1The pH was 5.8. Wherein TDZ is thidiazuron, and NAA is naphthylacetic acid.
S2, proliferation and strong seedling culture: and cutting the obtained adventitious bud cluster, inoculating the cut adventitious bud cluster into a strong seedling culture medium, and culturing the adventitious bud cluster into an adventitious bud. Adventitious buds cultured with strong seedlings are shown in FIG. 2.
Specifically, the adventitious bud cluster is cut into 3-5 buds/cluster or single bud, and the bud is inoculated into a strong seedling culture medium. After 2 months, strong adventitious buds are obtained and can be used for rooting culture.
The composition of the proliferation and strong seedling culture medium is as follows: WPM medium + TDZ 0-0.1 mg. L-1+IBA 0~0.1mg·L-1+GA3 5~7mg·L-1+ agar 7 g. L-1+ sucrose 30 g.L-1The pH was 5.8. Wherein IBA is indoleacetic acid (GA)3Is gibberellin.
TDZ and IBA can be added or not added in the strong seedling culture medium. TDZ and IBA are added into the culture medium, so that the adventitious bud can be further proliferated while the seedling is strong.
The following screening results of rhododendron lapponicum seedling strengthening culture medium are shown in the experimental example, rhododendron lapponicum is a large white rhododendron variety and is collected from a good seedling breeding base in the northern temperate zone of city, Chongqing city. The strong seedling culture medium is prepared by adding agar 7 g.L into WPM culture medium-1Sucrose (30 g. L)-1And adding the materials in the following formulas in the following table, wherein the specific experimental results are as follows:
TABLE 1 Rhododendron lapponicum seedling culture Medium screening orthogonal test results
In comparison 1 to comparison 3, TDZ added in a large amount exerts a certain inhibitory effect on the growth of adventitious buds.
S3, rooting culture: inoculating the adventitious bud after the strong seedling culture into a rooting culture medium, and culturing for 28-30 days; then taking out the adventitious bud and inoculating into a loose porous rooting matrix, culturing under aseptic condition, inducing to generate adventitious roots after 10-15 days, and growing into a rooted seedling with a complete root system after 50-60 days. FIG. 3 is a schematic diagram of rooting culture in a rooting medium; FIG. 4 is a rooted shoot with an intact root system formed by rooting culture.
The rooting medium comprises the following components: WPM culture medium and IBA 0.5-1 mg.L-1+ agar 7 g. L-1+ sucrose 30 g.L-1The pH was 5.8.
The following screening results of the alpine rhododendron rooting medium are shown in the experimental example, the alpine rhododendron is a rhododendron blanch variety and is collected from a good nursery stock breeding base in the northern temperate zone of city, Chongqing city. The strong seedling culture medium is prepared by adding agar 7 g.L into WPM culture medium-1Sucrose (30 g. L)-1And adding the materials in the following formulas in the following table, wherein the specific experimental results are as follows:
TABLE 2 Rhododendron lapponicum rooting medium screening test results
In comparative example 1, since IBA was added in excess, although adventitious shoots were still further extended and grown, they formed a large amount of callus at the basal part, indicating that IBA was used in excess, which was more favorable for callus induction and unfavorable for adventitious root formation.
Specifically, the rooting culture comprises two stages, namely, in the first stage, inoculating a strong adventitious bud with the length of about 2-3 cm into a rooting culture medium, and culturing for 28-30 days;
in the first stage, the nutrient in the culture medium is rich, the adventitious bud can fully absorb the nutrient in the culture medium, the nutrient requirement of the adventitious bud is higher and higher along with the growth of the adventitious bud, the nutrient in the culture medium is lower and lower, the nutrient gradually accumulates in the adventitious bud, and an adventitious root bulge is formed at the base part. Thus, during this culture process, the adventitious bud continuously absorbs and stores nutrients, which not only allows it to gain further growth, but also allows a simultaneous construction of a nutrient pool in its body.
And in the second stage, taking out the adventitious buds cultured in the first stage, inoculating the adventitious buds into a loose and porous rooting matrix, culturing for 10-15 days in a sterile environment of a conventional culture room to induce to generate adventitious roots, and growing into a rooted seedling with a complete root system in 50-60 days.
In the second stage, no nutrient is added, and because the adventitious buds have stored enough nutrients in vivo in the first stage, the adventitious buds cultured in the first stage are inoculated into a loose porous rooting matrix without any nutrients, and the adventitious buds can keep growing by using the nutrients stored in vivo. And meanwhile, the capacity of actively absorbing nutrition is further induced, the elongation growth of the adventitious bud is promoted to absorb nutrition in a poor nutrition environment to further meet the growth requirement, so that the adventitious bud gradually grows into a complete root system in a loose porous rooting matrix without any nutrient substances, the rooting culture of the adventitious bud is realized, and a rooted seedling is formed.
The loose porous rooting matrix is a sterile matrix which can be loose porous perlite and/or loose porous vermiculite.
Specifically, the mass-to-volume ratio of the loose porous matrix to water is 1: 2.5-3, in the second stage of rooting culture, sufficient water is added according to the proportion of the matrix to the water, the growth requirement of the adventitious bud is guaranteed, the water in the matrix is gradually reduced along with the extension of the culture time, the development of the adventitious bud root primordium is further induced, the elongation growth of the adventitious root is promoted, and the water is actively absorbed. When the culture is carried out for about 40 days, certain water can be added according to actual conditions, so that the loose porous rooting matrix is kept in a moist state suitable for rooting, and the adventitious bud is prevented from dying due to drought.
The rooting culture can be carried out in a conventional culture room, and specifically, the culture environment is as follows: the illumination time is 16h d-1Dark culture is 8 h.d-1The light intensity is 50 mu mol.m-2·s-1The temperature is (25 +/-2) DEG C.
During the rooting culture of the second stage, NAA with a certain concentration can be added into the rooting matrix according to the difference of rooting difficulty of the seeds or the varieties, and the addition amount of the NAA is 0-0.5 mg.L-1To promote the formation of adventitious roots.
The rooting method fully utilizes the existing sterile environment of a culture room, induces the adventitious bud to generate a complete root system under the sterile condition, is less influenced by the environment and can be implemented all year round.
S4, transplanting rooting seedlings: transplanting the rooted seedlings to obtain survival rhododendron lapponicum tissue culture seedlings. The tissue culture seedlings transplanted to survive are shown in FIG. 5.
When the rooted seedlings are transplanted, transplanting the rooted seedlings with complete root systems into a seedling hardening transplanting substrate, wherein the substrate is formed by mixing turf and perlite, the pH value of the substrate is 5.9-7.0, and after the rooted seedlings are cultured for 30-40 days, the survival rhododendron lapponicum tissue culture seedlings are obtained.
The ratio of the turf to the perlite in the matrix is 1:1 or 1:2.
After transplanting, the rooted seedlings are placed in a shady and ventilated environment with the relative air humidity not lower than 80%, and can survive after 1 month.
The application provides a rhododendron alpinum tissue culture propagation method, after an explant of the rhododendron alpinum is subjected to induction, proliferation and strong seedling culture to form a stout adventitious bud, the adventitious bud is inoculated in a rooting culture medium for rooting culture, and the adventitious bud is cultured for 28-30 days to fully absorb nutrition. Meanwhile, the adventitious bud can grow further in the rooting culture medium, and a nutrient bank is synchronously constructed in the body of the adventitious bud to store nutrient substances; then taking out the adventitious bud, placing the adventitious bud in a loose porous rooting matrix, culturing under an aseptic condition, culturing a complete root system with an active nutrition absorption function under the aseptic condition by utilizing the improved rooting matrix with air permeability and combining stock nutrition in the adventitious bud, improving the growth potential of the tissue culture seedling, enhancing the resistance of the tissue culture seedling to environmental stress in the seedling exercising and transplanting process, improving the transplanting survival rate, and solving the problems of slow rooting outside a tissue culture seedling bottle, long seedling exercising and transplanting time, low survival rate and the like in the large-scale production of the specific variety of rhododendron lapponicum.
In the tissue culture propagation process, the culture material is always in an aseptic environment, and the tissue culture seedling can not move out of the aseptic environment until the tissue culture seedling is acclimatized and transplanted. The whole tissue culture steps are few, the flow is simple, the addition of various additives and the like is effectively reduced, the self nutrition is fully utilized to achieve the rooting effect, the cost is saved, the energy consumption is reduced, and the tissue culture method has the characteristics of environmental friendliness, strong practicability and applicability and the like.
While particular embodiments of the present invention have been illustrated and described, it will be appreciated that the present invention is not limited to the above-described alternative embodiments, and that various other forms of product may be devised by anyone in light of the present invention. The foregoing detailed description should not be construed as limiting the scope of the invention, and persons skilled in the art should understand that changes may be made in the method solutions described in the foregoing embodiments, or equivalent substitutions may be made in part or all of the method features without departing from the spirit and scope of the invention, and that such changes or substitutions do not substantially depart from the essence of the corresponding method solutions.

Claims (10)

1. A tissue culture propagation method of rhododendron lapponicum comprises the following steps:
s1, explant induction: inoculating a sterile explant of rhododendron lapponicum into an induction culture medium, and inducing to generate an adventitious bud cluster;
s2, proliferation and strong seedling culture: cutting the generated adventitious bud cluster, inoculating the cut adventitious bud cluster into a strong seedling culture medium, and culturing the adventitious bud cluster into adventitious buds;
it is characterized by also comprising the following steps,
s3, rooting culture: inoculating the adventitious bud after the strong seedling culture into a rooting culture medium, and culturing for 28-30 days; then taking out the adventitious bud and inoculating the adventitious bud into a loose porous rooting matrix, culturing under an aseptic condition, inducing to generate adventitious roots after 10-15 days, and growing into a rooting seedling with a complete root system after 50-60 days;
s4, transplanting rooting seedlings: transplanting the rooted seedlings to obtain survival rhododendron lapponicum tissue culture seedlings.
2. The tissue culture propagation method of rhododendron lapponicum according to claim 1, wherein the sterile explant is a bud or/and a terminal bud in a bud of rhododendron lapponicum.
3. The tissue culture propagation method of rhododendron lapponicum according to claim 2, wherein the sterile explant is obtained by the following method:
selecting a flower bud which has a length of 4-5 cm, is not expanded and blooms and is kept in a perfect aseptic environment inside, spraying an ethanol solution on the surface of the flower bud for sterilization, and circularly peeling bracts of the flower bud layer by using an aseptic blade in the aseptic environment to obtain an aseptic flower bud or/and an aseptic terminal bud, wherein the aseptic flower bud or/and the aseptic terminal bud are aseptic explants.
4. The tissue culture propagation method of rhododendron lapponicum according to claim 1, wherein in step S1, the non-contaminated explants are cut 10-14 days after the sterile explants are inoculated into the induction medium, and then are inoculated into the induction medium again, and after 80-90 days, callus is obtained and adventitious bud clusters are formed.
5. The tissue culture propagation method of alpine rhododendron, according to claim 1, characterized in that in step S4, when the rooted seedlings are transplanted, the rooted seedlings with complete root systems are transplanted into a growth substrate which is obtained by mixing turf and perlite and has a pH value of 5.9-7.0, and the survival alpine rhododendron tissue culture seedlings are obtained after 30-40 days of culture.
6. The tissue culture propagation method of rhododendron lapponicum according to claim 1, wherein in S1, the induction medium consists of WPM medium + TDZ + NAA + agar + sucrose;
the mass concentration of the TDZ is 0.5-2 mg.L-1(ii) a The mass concentration of NAA is 0-0.5 mg.L-1(ii) a The mass concentration of agar is 7 g.L-1(ii) a The mass concentration of sucrose is 30 g.L-1
The pH of the induction medium was 5.8.
7. The tissue culture propagation method of rhododendron lapponicum according to claim 1, wherein in S2, the culture medium for propagation and strong seedling consists of WPM culture medium + TDZ + IBA + GA3+ agar + sucrose;
the mass concentration of the TDZ is 0-0.1 mg.L-1(ii) a The mass concentration of IBA is 0-0.1 mg.L-1;GA3Has a mass concentration of 5 to 7 mg.L-1(ii) a The mass concentration of agar is 7 g.L-1(ii) a The mass concentration of sucrose is 30 g.L-1
The pH value of the proliferation and strong seedling culture medium is 5.8.
8. The tissue culture propagation method of rhododendron lapponicum according to claim 1, wherein in S3, the rooting medium is composed of WPM medium + IBA + agar + sucrose,
the mass concentration of IBA is 0.5-1 mg.L-1(ii) a The mass concentration of agar is 7 g.L-1(ii) a The mass concentration of sucrose is 30 g.L-1
The pH value of the rooting medium is 5.8.
9. The tissue culture propagation method of rhododendron lapponicum according to claim 1, wherein in the step S3, the loose porous rooting matrix is loose porous perlite and/or loose porous vermiculite.
10. The tissue culture propagation method of rhododendron lapponicum according to claim 1, wherein in S4, when the rooted seedlings are transplanted, the rooted seedlings are moved out of a sterile environment and cultured in a cool and ventilated place with the relative air humidity not lower than 80%.
CN202110425793.1A 2021-04-20 2021-04-20 Tissue culture propagation method for alpine rhododendron Pending CN113100060A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202110425793.1A CN113100060A (en) 2021-04-20 2021-04-20 Tissue culture propagation method for alpine rhododendron

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202110425793.1A CN113100060A (en) 2021-04-20 2021-04-20 Tissue culture propagation method for alpine rhododendron

Publications (1)

Publication Number Publication Date
CN113100060A true CN113100060A (en) 2021-07-13

Family

ID=76719474

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202110425793.1A Pending CN113100060A (en) 2021-04-20 2021-04-20 Tissue culture propagation method for alpine rhododendron

Country Status (1)

Country Link
CN (1) CN113100060A (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113383652A (en) * 2021-07-19 2021-09-14 石家庄市农林科学研究院 Technical method for inducing lateral roots of alpine rhododendron
CN113767848A (en) * 2021-08-25 2021-12-10 山东省农业科学院 Efficient rooting method for rhododendron lapponicum tissue culture seedlings

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH06189643A (en) * 1992-12-25 1994-07-12 New Oji Paper Co Ltd Large-scale multiplication of rhododendron
CN103636492A (en) * 2013-11-14 2014-03-19 石家庄市农林科学研究院 Rhododendron hybrides cosmopolitan tissue culture rapid propagation method

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH06189643A (en) * 1992-12-25 1994-07-12 New Oji Paper Co Ltd Large-scale multiplication of rhododendron
CN103636492A (en) * 2013-11-14 2014-03-19 石家庄市农林科学研究院 Rhododendron hybrides cosmopolitan tissue culture rapid propagation method

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
刘晓青等: "高山杜鹃茎段组织培养和优化体系的建立", 《扬州大学学报(农业与生命科学版)》 *
汤桂钧等: "高山杜鹃的组织培养快速繁殖技术研究", 《上海农业学报》 *
管耀义等: "高山杜鹃叶片再生植株的研究", 《河北林业科技》 *
苏家乐等: "高山杜鹃新品种富丽金陵的组培快繁技术", 《江苏农业科学》 *
郭颖: "三种高山杜鹃组织培养快繁技术研究", 《中国优秀硕士学位论文全文数据库 农业科级辑》 *
黄萍萍: "高山杜鹃(蒙他瑰)快速繁殖技术研究", 《福建农业科技》 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113383652A (en) * 2021-07-19 2021-09-14 石家庄市农林科学研究院 Technical method for inducing lateral roots of alpine rhododendron
CN113767848A (en) * 2021-08-25 2021-12-10 山东省农业科学院 Efficient rooting method for rhododendron lapponicum tissue culture seedlings

Similar Documents

Publication Publication Date Title
CN101366357B (en) Method for tissue culture and quick propagation technique of reddish blue spider lily
CN113100060A (en) Tissue culture propagation method for alpine rhododendron
CN105815213A (en) Establishing method for in-vitro regeneration system of Kiwi berry
CN102845313A (en) Method for quickly in-vitro actinidia kolomikta propagating
CN103931497A (en) Method for improving seedling rate of tissue culture seedlings of hylocereus undulatus britt
CN102823502A (en) Method for intermediately propagating and culturing vitis quinquangularis in vitro
CN100391333C (en) Aseptic seedling tissue culturing and test tube seedling hardening off and transplating technology for anthurium andraeanum
CN102210267B (en) Method for regenerating rose into complete plant
CN110547196B (en) Method for quickly propagating air pineapple seedlings through leaf tissue culture
CN100429306C (en) Tissue culture medium and fast propagation method for Sorbone lily
CN103155869A (en) Sweet cherry rootstock Colt tissue culture method
CN101743908A (en) Tissue culture, rapid propagation and cultivation method of grevillea banksii
CN101637123B (en) Organic-culturing quick-reproducing method of curcuma
CN108782247A (en) A kind of method for tissue culture of late cherry " Yu Yihuang " kind of Japan
CN106879473B (en) A kind of method that larch Vitro Plant occurs
CN1631105A (en) Clonal propagation method of Momordica grosvenori germchit
CN107484665A (en) A kind of method using black fruit fructus lycii resting shoot seedling
CN104126505A (en) Somatic embryo in-vitro regeneration method applied to genetic transformation and seed ball rapid propagation of Lilium pumilumDC. Fisch
JP4687028B2 (en) Cutting plant seedling production method of Eucalyptus plant
CN101946704A (en) Method for regenerating Chinese rose plant by using immature seed as explant
CN103609444A (en) Tissue culture method for hemerocallis sempervirens araki
CN112042540A (en) Rapid propagation method of hydrangea macrophylla
CN102763598B (en) Method for breeding wenshan paphiopedilum seedlings by using somatic embryo
CN108064690B (en) Tissue culture method of herba Amaranthi Tricoloris
CN108934713A (en) A kind of acer catalpifolium seed seedling-raising method

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