CN112931224A - Tissue culture method of morinda officinalis - Google Patents
Tissue culture method of morinda officinalis Download PDFInfo
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- CN112931224A CN112931224A CN202110416388.3A CN202110416388A CN112931224A CN 112931224 A CN112931224 A CN 112931224A CN 202110416388 A CN202110416388 A CN 202110416388A CN 112931224 A CN112931224 A CN 112931224A
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
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- 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
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Abstract
The invention relates to the technical field of tissue culture, in particular to a tissue culture method of morinda officinalis, which comprises the steps of taking disinfected and cleaned morinda officinalis rattan as an explant; inoculating the explant to an axillary bud induction culture medium, and illuminating every day until the axillary bud grows out; inoculating axillary buds to a cluster bud induction culture medium, and illuminating every day until cluster buds grow out; and (4) inoculating the cluster buds to a rooting induction culture medium, and illuminating every day until a root system grows out. The tissue culture method of morinda officinalis disclosed by the invention adopts the rattan of morinda officinalis as an explant to carry out tissue culture, namely a direct organ generation way of morinda officinalis is adopted, a callus stage is not needed, the morinda officinalis has good genetic stability, variation is not easy to occur, and the specific medicinal properties of morinda officinalis can be effectively maintained.
Description
Technical Field
The invention relates to the technical field of tissue culture, in particular to a tissue culture method of morinda officinalis.
Background
Morinda officinalis (Morinda officinalis How) is a plant of Rubiaceae (Rubiaceae), one of the well-known "four great south drugs". The dried root of the Chinese medicinal composition has the effects of strengthening bones and muscles, nourishing kidney yang, dispelling wind and removing dampness. Meanwhile, the morinda officinalis has a wide health care effect and can be widely used in preserved fruits, soup bases, food materials and the like. In recent years, the demand of morinda officinalis has increased gradually; at present, morinda officinalis is cultivated and planted mainly through asexual propagation methods such as cutting propagation, the method is simple and high in survival rate, but the asexual propagation creates conditions for virus accumulation, and the germplasm of morinda officinalis is degraded, more diseases are caused, the yield is reduced, and the quality is reduced.
In the related art, in order to solve the above problems, an indirect organogenesis approach is adopted for tissue culture of morinda officinalis, a callus is induced by a morinda officinalis explant, and adventitious buds are induced by the callus to obtain a tissue culture seedling of morinda officinalis; however, the method of obtaining the tissue culture seedling of morinda officinalis through callus induction adventitious buds has poor genetic stability, is easy to generate variation, and is not beneficial to keeping the specific characters of medicinal plants.
Disclosure of Invention
The method adopts the rattan of the morinda officinalis as an explant to perform tissue culture, namely a direct organ generation way of the morinda officinalis is adopted, a callus stage is not needed, the morinda officinalis has good genetic stability, variation is not easy to occur, and the specific medicinal properties of the morinda officinalis can be effectively maintained.
The invention is realized by the following steps:
in a first aspect, the present invention provides a method for tissue culture of morinda officinalis, comprising:
taking the sterilized and cleaned morinda officinalis rattan as an explant;
inoculating the explant to an axillary bud induction culture medium, and illuminating every day until the axillary bud grows out;
inoculating axillary buds to a cluster bud induction culture medium, and illuminating every day until cluster buds grow out;
and (4) inoculating the cluster buds to a rooting induction culture medium, and illuminating every day until a root system grows out.
In an alternative embodiment, when the explant is inoculated in the axillary bud induction medium, the intensity of light per day is 1100-1300lx, and the duration of light per day is 11-13 h.
In an alternative embodiment, when the axillary bud is inoculated on the cluster bud induction medium, the intensity of light irradiation is 1100-1300lx per day, and the time period of light irradiation is 11-13h per day.
In an alternative embodiment, the intensity of light per day is 1100-1300lx and the duration of light per day is 11-13h when the cluster buds are inoculated into the rooting induction medium.
In an alternative embodiment, the axillary bud induction medium is 1/2MS +0.2 mg/L6-BA medium.
In an alternative embodiment, the cluster shoot induction medium is 1/2MS +0.2 mg/L6-BA medium.
In an alternative embodiment, the rooting induction medium is 1/2MS +0.5mg/L IBA medium.
In alternative embodiments, the explant comprises young shoot segments, semi-lignified shoot segments, and fully lignified shoot segments.
In an alternative embodiment, the method of tissue culture of morinda citrifolia further comprises explant conditioning, the explant conditioning comprising: transplanting Morinda officinalis plant to greenhouse, removing rattan, spraying carbendazim solution at intervals, and allowing new rattan to grow out as explant.
In an optional embodiment, the carbendazim solution has a mass concentration of 8% -12%, and is sprayed once every 5-8 days.
The invention has the following beneficial effects:
the tissue culture method of morinda officinalis comprises taking disinfected and cleaned morinda officinalis rattan as an explant; inoculating the explant to an axillary bud induction culture medium, and illuminating every day until the axillary bud grows out; inoculating axillary buds to a cluster bud induction culture medium, and illuminating every day until cluster buds grow out; and (4) inoculating the cluster buds to a rooting induction culture medium, and illuminating every day until a root system grows out. Therefore, the morinda officinalis tissue culture method provided by the invention can utilize the rattan of morinda officinalis as an explant to perform a direct organogenesis way without inducing and differentiating callus, does not pass through the callus, is favorable for good genetic stability, is not easy to mutate, and can effectively maintain the specific medicinal properties of morinda officinalis.
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In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
FIG. 1 is a schematic representation of a new growing cane in example 3 of the present invention;
FIG. 2 is a diagram showing the growth of axillary buds induced in example 3 of the present invention;
FIG. 3 is a schematic representation showing the growth of multiple shoots in example 3 of the present invention;
FIG. 4 is a diagram showing the condition of inducing rooting in example 3 of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The features and properties of the present invention are described in further detail below with reference to examples.
The invention provides a tissue culture method of morinda officinalis, which comprises the following steps: taking the sterilized and cleaned morinda officinalis rattan as an explant; inoculating the explant to an axillary bud induction culture medium, and illuminating every day until the axillary bud grows out; inoculating axillary buds to a cluster bud induction culture medium, and illuminating every day until cluster buds grow out; and (4) inoculating the cluster buds to a rooting induction culture medium, and illuminating every day until a root system grows out. Therefore, the morinda officinalis tissue culture method provided by the invention can utilize the rattan of morinda officinalis as an explant to perform a direct organogenesis way without inducing and differentiating callus, does not pass through the callus, is favorable for good genetic stability, is not easy to mutate, and can effectively maintain the specific medicinal properties of morinda officinalis.
Tissue culture through an organogenesis approach is a main mode of medicinal plant tissue culture, and refers to the steps of inducing an explant to generate organs such as adventitious buds and the like under certain conditions, and performing propagation, root induction, hardening seedlings, transplanting and the like to develop a complete plant. Through the indirect organogenesis approach of callus, not only variation is easy to occur, which causes great difference in the regeneration quality of medicinal plants, but also the problems of long culture time, low propagation coefficient, difficult proliferation and long successive growth period exist. The method does not pass through a direct organ generation way of callus, and can quickly obtain axillary buds and other direct organs without inducing and differentiating the callus, thereby effectively shortening the cycle time, increasing the propagation coefficient, reducing the propagation difficulty and the successive transfer growth cycle.
The tissue culture method of morinda officinalis further comprises explant pretreatment, wherein the explant pretreatment comprises the following steps: transplanting Morinda officinalis plant to greenhouse, removing rattan, spraying carbendazim solution at intervals, and allowing new rattan to grow out as explant.
Optionally, selecting adult morinda officinalis plants which are 3-4 years old, transplanting the morinda officinalis plants into a clean greenhouse, cutting off rattan from the stem base of morinda officinalis, and spraying carbendazim solution to the transplanted morinda officinalis plants every 5-8 days, wherein the mass concentration of the carbendazim solution is 8% -12%.
It should be noted that, the transplanted morinda officinalis usually grows new rattan after one month, and the new rattan can be used as the explant for tissue culture.
Removing new rattan from transplanted Morinda officinalis plant, removing leaves, placing the stem on a clean bench, wiping the surface of the stem with 70-80% alcohol, and cutting the wiped stem into stem segments with nodes and length of 2-3 cm; wherein, the stem segment can be divided into a tender stem segment, a semi-lignified stem segment and a full-lignified stem segment, namely, the tender stem segment, the semi-lignified stem segment and the full-lignified stem segment can be used as explants for tissue culture.
It should be noted that the young stem is a part within about 20cm from the top end, the semi-lignified stem is a stem about 20-40cm from the top end, and the fully-lignified stem is a stem about more than 40cm from the top end.
It should be further noted that both leaves and stem segments can be used as explants for tissue culture, and the differentiation capacity of each explant is different, wherein the semi-lignified stem segment has stronger differentiation capacity and good application value in the tissue culture of morinda officinalis.
Because the stem sections at all the parts have different surface roughness and different bacterium carrying capacity, different disinfection modes can be adopted aiming at different stem sections so as to ensure that the stem sections at all the parts obtain good disinfection effect.
Optionally, the young and tender stem segments of Morinda officinalis can be sterilized with 0.05% -0.1% mercuric chloride solution for about 4-5 min; multiple rinses with sterile water may be used after sterilization, for example: rinsing 3 times, 4 times, 5 times, etc., and is not particularly limited herein.
The semi-lignified stem segment and the full-lignified stem segment can be disinfected by mercuric chloride solution with the mass concentration of 0.05-0.1% for about 5-6min, and can be rinsed by sterile water for multiple times after disinfection. Further, the semi-lignified stem segment and the full-lignified stem segment can be sterilized twice by using a mercuric chloride solution with the mass concentration of 0.05% -0.1%, each time lasts for about 2.5-3min, and after each sterilization, the stem segments can be rinsed for multiple times by using sterile water, for example: rinsing 3 times, 4 times, 5 times, etc., and is not particularly limited herein.
After each stem section is disinfected and cleaned, inoculation can be carried out. Specifically, the explants are inoculated in the axillary bud induction medium under the aseptic condition, and the culture medium is irradiated for 11-13h every day, and the intensity of the irradiation is 1100-1300lx every day.
Further, in the process of inducing axillary bud differentiation, the air temperature can be 27-29 ℃ and the humidity can be 70-80%.
The axillary bud induction medium can be selected according to needs, and is 1/2MS +0.2 mg/L6-BA medium; specifically, the axillary bud induction medium is: 1/2MS +6-BA0.2mg/L +30g/L sucrose +4.3g/L agar powder, and the pH is 6.0.
It should be further noted that axillary buds can grow after the axillary buds are induced and cultured for about 30 days.
After the axillary bud is induced, cluster buds need to be induced, specifically, the axillary buds cultured according to the method are cut off from the cultured morinda officinalis stem segments and inoculated in a cluster bud induction culture medium, the illumination is carried out for 11-13h every day, and the illumination intensity is 1100-1300lx every day.
Further, in the process of inducing the cluster buds, the air temperature is 27-29 ℃, and the humidity is 70-80%.
The cluster bud induction medium can be selected according to needs, and is 1/2MS +0.2 mg/L6-BA medium, and the induction rate of the cluster bud can approximately reach about 86.36%; specifically, the culture medium for inducing the cluster buds is as follows: 1/2MS +6-BA0.2mg/L +30g/L sucrose +4.3g/L agar powder, and the pH is 6.0.
It should be further noted that the cluster buds can grow out after about 30 days of induced culture.
After the cluster buds are induced, rooting needs to be induced, specifically, the cluster buds cultured according to the method are cut off from the stem segments of the cultured morinda officinalis and inoculated in a rooting induction culture medium, and the illumination is 11-13h every day, and the illumination intensity is 1100-.
Further, in the process of inducing rooting, the air temperature is 27-29 ℃, and the humidity is 70-80%.
It should be noted that the rooting induction medium can be selected according to the needs, and the rooting induction medium in this embodiment is 1/2MS +0.5mg/L IBA medium; specifically, the rooting induction culture medium comprises: 1/2MS + IBA0.5mg/L +30g/L sucrose +4.3g/L agar powder, and the PH is 6.0.
It should be further explained that the root system can grow out after approximately 30 days of rooting induction culture.
It is noted that when axillary buds, cluster buds and roots are induced, the axillary buds, the cluster buds and the roots are irradiated for a period of time under certain intensity of illumination, so that the seedling exercising effect is good, tissue seedlings cultured by the tissue culture can adapt to subsequent transplantation more quickly, and the survival rate is improved.
Hereinafter, the tissue culture method of morinda officinalis according to the present invention will be described in detail with reference to examples.
Example 1
Taking sterilized and cleaned morinda officinalis rattan as an explant, and specifically, taking the explant as a semi-lignified stem section.
Inoculating the explant to an axillary bud induction culture medium, irradiating for 11h every day, wherein the illumination intensity is 1300lx every day, the air temperature is 28 ℃, the humidity is 75%, and the axillary bud grows out after culturing for 30 days. Wherein the axillary bud induction culture medium is 1/2MS +6-BA0.2mg/L +30g/L sucrose +4.3g/L agar powder, and the pH is 6.0.
Inoculating axillary buds to a cluster bud induction culture medium, illuminating for 13h every day, wherein the illumination intensity is 1200lx every day, the air temperature is 28 ℃, the humidity is 75%, and after culturing for 30 days, cluster buds grow out. Wherein the cluster bud induction culture medium is 1/2MS +6-BA0.2mg/L +30g/L sucrose +4.3g/L agar powder, and the pH is 6.0.
And (3) inoculating the cluster buds to a rooting induction culture medium, illuminating for 12h each day, wherein the illumination intensity is 1200lx each day, the air temperature is 28 ℃, the humidity is 77%, and the root system grows out after culturing for 30 days. Wherein, the rooting induction culture medium is as follows: 1/2MS + IBA0.5mg/L +30g/L sucrose +4.3g/L agar powder, and the PH is 6.0.
Example 2
Pretreating an explant, selecting an adult morinda officinalis which grows for 3-4 years, transplanting the adult morinda officinalis into a clean greenhouse, cutting off rattan from the base of the morinda officinalis stem, spraying a carbendazim solution with the mass concentration of 10% to the morinda officinalis every 7 days, and growing a new rattan as an explant material after one month.
Sterilizing and cleaning the explants; the explant comprises three types of tender parts, semi-lignified stem segments and fully-lignified stem segments.
Inoculating the explant to an axillary bud induction culture medium, irradiating for 12h each day with illumination intensity of 1200lx each day, air temperature of 28 ℃ and humidity of 75%, and culturing for 30 days to allow axillary buds to grow out. Wherein the axillary bud induction culture medium is 1/2MS +6-BA0.2mg/L +30g/L sucrose +4.3g/L agar powder, and the pH is 6.0.
Inoculating axillary buds to a cluster bud induction culture medium, illuminating for 12h each day with illumination intensity of 1200lx each day, air temperature of 28 ℃, humidity of 75%, and culturing for 30 days to grow cluster buds. Wherein the cluster bud induction culture medium is 1/2MS +6-BA0.2mg/L +30g/L sucrose +4.3g/L agar powder, and the pH is 6.0.
And (3) inoculating the cluster buds to a rooting induction culture medium, illuminating for 12h each day, wherein the illumination intensity is 1200lx each day, the air temperature is 28 ℃, the humidity is 75%, and the roots grow out after culturing for 30 days. Wherein, the rooting induction culture medium is as follows: 1/2MS + IBA0.5mg/L +30g/L sucrose +4.3g/L agar powder, and the PH is 6.0.
Example 3
Pretreating explants, selecting 3-4 years old morinda officinalis, transplanting into a clean greenhouse, cutting off rattans from the stem base of morinda officinalis, spraying carbendazim solution with the mass concentration of 10% to morinda officinalis every 7 days, and growing new rattans as explant materials after one month (as shown in figure 1).
Placing the new growing rattan explant material on a clean bench, wiping the surface of the explant material with 75% alcohol, and cutting into stem segments with nodes and length of 2-3cm, wherein the cut stem segments comprise young stem segments within 20cm from the top end, semi-lignified stem segments within 20-40cm from the top end, and fully lignified stem segments exceeding 40cm from the top end. Sterilizing young and tender stem segments with 0.1% mercuric chloride solution for 4min30s, and sterilizing semi-lignified and fully lignified stem segments with 0.1% mercuric chloride solution for 6 min; after sterilization, each stem section was rinsed with sterile water several times to obtain explants for inoculation.
Inoculating the explant to axillary bud induction culture medium, irradiating with light for 12 hr per day at illumination intensity of 1200lx, air temperature of 28 deg.C and humidity of 75%, and culturing for 30 days to obtain axillary bud (shown in FIG. 2). Wherein the axillary bud induction culture medium is 1/2MS +6-BA0.2mg/L +30g/L sucrose +4.3g/L agar powder, and the pH is 6.0.
Inoculating axillary buds to cluster bud induction culture medium, irradiating with light of 1200lx, air temperature of 28 deg.C, humidity of 75% for 12 hr per day, and culturing for 30 days to grow cluster buds (as shown in FIG. 3). Wherein the cluster bud induction culture medium is 1/2MS +6-BA0.2mg/L +30g/L sucrose +4.3g/L agar powder, and the pH is 6.0.
The cluster buds are inoculated in a rooting induction culture medium, the illumination is 12 hours per day, the illumination intensity is 1200lx per day, the air temperature is 28 ℃, the humidity is 75 percent, and the root system grows out after the culture is carried out for 30 days (as shown in figure 4). Wherein, the rooting induction culture medium is as follows: 1/2MS + IBA0.5mg/L +30g/L sucrose +4.3g/L agar powder, and the PH is 6.0.
Example 4
Pretreating an explant, selecting a 3-year-old morinda officinalis from an adult plant, transplanting the morinda officinalis into a clean greenhouse, cutting off rattan from the base of a morinda officinalis stem, spraying a carbendazim solution with the mass concentration of 8% to the morinda officinalis every 5 days, and growing a new rattan as an explant material after one month.
Placing the newly grown rattan explant material on a clean bench, wiping the surface of the explant material with 70% alcohol, and cutting into stem segments with nodes and about 2cm in length, wherein the cut stem segments comprise young stem segments within 20cm from the top end, semi-lignified stem segments within 20-40cm from the top end and fully-lignified stem segments within 40cm from the top end. Sterilizing young and tender stem segments with 0.1% mercuric chloride solution for 4min, sterilizing semi-lignified and fully-lignified stem segments with 0.1% mercuric chloride solution for two times, 3min each time, and rinsing with sterile water after each sterilization; after sterilization, each stem section was rinsed with sterile water several times to obtain explants for inoculation.
Inoculating the explant to an axillary bud induction culture medium, irradiating for 11h each day, wherein the illumination intensity is 1300lx each day, the air temperature is 27.2 ℃, the humidity is 72%, and the axillary bud grows out after culturing for 30 days. Wherein the axillary bud induction culture medium is 1/2MS +6-BA0.2mg/L +30g/L sucrose +4.3g/L agar powder, and the pH is 6.0.
Inoculating axillary buds to a cluster bud induction culture medium, and illuminating for 13h every day, wherein the illumination intensity is 1100lx every day, the air temperature is 27 ℃, the humidity is 78%, and the cluster buds grow out after culturing for 30 days. Wherein the cluster bud induction culture medium is 1/2MS +6-BA0.2mg/L +30g/L sucrose +4.3g/L agar powder, and the pH is 6.0.
And (3) inoculating the cluster buds to a rooting induction culture medium, illuminating for 11h every day, wherein the illumination intensity is 1100lx every day, the air temperature is 27.1 ℃, the humidity is 72%, and the root system grows out after culturing for 30 days. Wherein, the rooting induction culture medium is as follows: 1/2MS + IBA0.5mg/L +30g/L sucrose +4.3g/L agar powder, and the PH is 6.0.
Example 5
Pretreating an explant, selecting 4-year-old morinda officinalis, transplanting the morinda officinalis into a clean greenhouse, cutting off rattan from the base of morinda officinalis stem, spraying carbendazim solution with the mass concentration of 12% to morinda officinalis every 8 days, and growing new rattan as an explant material after one month.
Placing the newly grown rattan explant material on a clean bench, wiping the surface of the explant material with 80% alcohol, and cutting into stem segments with nodes and about 3cm in length, wherein the cut stem segments comprise young stem segments within 20cm from the top end, semi-lignified stem segments within 20-40cm from the top end and fully-lignified stem segments within 40cm from the top end. Sterilizing young and tender stem segments with 0.05% mercuric chloride solution for 5mins, and sterilizing semi-lignified and full-lignified stem segments with 0.05% mercuric chloride solution for 6 min; after sterilization, each stem section was rinsed with sterile water several times to obtain explants for inoculation.
Inoculating the explant to an axillary bud induction culture medium, and irradiating for 13h every day, wherein the illumination intensity every day is 1100lx, the air temperature is 29 ℃, the humidity is 80%, and the axillary bud grows out after culturing for 30 days. Wherein the axillary bud induction culture medium is 1/2MS +6-BA0.2mg/L +30g/L sucrose +4.3g/L agar powder, and the pH is 6.0.
Inoculating axillary buds to a cluster bud induction culture medium, illuminating for 11h every day, wherein the illumination intensity is 1300lx every day, the air temperature is 29 ℃, the humidity is 70%, and the cluster buds grow out after culturing for 30 days. Wherein the cluster bud induction culture medium is 1/2MS +6-BA0.2mg/L +30g/L sucrose +4.3g/L agar powder, and the pH is 6.0.
And (3) inoculating the cluster buds to a rooting induction culture medium, and irradiating for 13h every day with the illumination intensity of 1300lx, the air temperature of 28.8 ℃ and the humidity of 79%, wherein the root system grows out after culturing for 30 days. Wherein, the rooting induction culture medium is as follows: 1/2MS + IBA0.5mg/L +30g/L sucrose +4.3g/L agar powder, and the PH is 6.0.
Example 6
Pretreating an explant, selecting an adult morinda officinalis which grows for 3-4 years, transplanting the adult morinda officinalis into a clean greenhouse, cutting off rattan from the base of the morinda officinalis stem, spraying a carbendazim solution with the mass concentration of 11% to the morinda officinalis every 6 days, and growing a new rattan as an explant material after one month.
Placing the new growing rattan explant material on a clean bench, wiping the surface of the explant material with 75% alcohol, and cutting into stem segments with nodes and length of 2-3cm, wherein the cut stem segments comprise young stem segments within 20cm from the top end, semi-lignified stem segments within 20-40cm from the top end, and fully lignified stem segments exceeding 40cm from the top end. Sterilizing young and tender stem segments with 0.08% mercuric chloride solution for 4min30s, sterilizing semi-lignified and fully lignified stem segments with 0.08% mercuric chloride solution for 3min twice, and rinsing with sterile water after each sterilization; after sterilization, each stem section was rinsed with sterile water several times to obtain explants for inoculation.
Inoculating the explant to an axillary bud induction culture medium, irradiating for 12h each day, wherein the illumination intensity is 1200lx each day, the air temperature is 28.3 ℃, the humidity is 75%, and axillary buds grow out after culturing for 30 days. Wherein the axillary bud induction culture medium is 1/2MS +6-BA0.2mg/L +30g/L sucrose +4.3g/L agar powder, and the pH is 6.0.
Inoculating axillary buds to a cluster bud induction culture medium, illuminating for 12h each day, wherein the illumination intensity is 1200lx each day, the air temperature is 27.8 ℃, the humidity is 75%, and the cluster buds grow out after culturing for 30 days. Wherein the cluster bud induction culture medium is 1/2MS +6-BA0.2mg/L +30g/L sucrose +4.3g/L agar powder, and the pH is 6.0.
And (3) inoculating the cluster buds to a rooting induction culture medium, illuminating for 12h each day, wherein the illumination intensity is 1200lx each day, the air temperature is 28.5 ℃, the humidity is 75%, and the root system grows out after culturing for 30 days. Wherein, the rooting induction culture medium is as follows: 1/2MS + IBA0.5mg/L +30g/L sucrose +4.3g/L agar powder, and the PH is 6.0.
In conclusion, the tissue culture method of morinda officinalis provided by the invention adopts the rattan of morinda officinalis as the explant for inoculation culture, callus does not need to be induced and differentiated, good genetic stability is facilitated, variation is not easy to occur, specific medicinal properties of morinda officinalis can be effectively maintained, and the culture period can be shortened.
The above is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and various modifications and changes will occur to those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A tissue culture method of Morinda officinalis, comprising:
taking the sterilized and cleaned morinda officinalis rattan as an explant;
inoculating the explant to an axillary bud induction culture medium, and illuminating every day until the axillary bud grows out;
inoculating the axillary buds to a cluster bud induction culture medium, and illuminating every day until cluster buds grow out;
and (4) inoculating the cluster buds to a rooting induction culture medium, and illuminating every day until a root system grows out.
2. The method of claim 1, wherein the intensity of light exposure is 1100-.
3. The method of claim 1, wherein the axillary bud is inoculated on the cluster bud induction medium at a light intensity of 1100-1300lx per day for a period of 11-13 h.
4. The tissue culture method of Morinda citrifolia according to claim 1, wherein the intensity of light is 1100-.
5. The tissue culture method of morinda officinalis how of claim 1, wherein said axillary bud induction medium is 1/2MS +0.2 mg/L6-BA medium.
6. The tissue culture method of morinda officinalis how of claim 1, wherein said multiple shoot induction medium is 1/2MS +0.2 mg/L6-BA medium.
7. The tissue culture method of morinda officinalis how of claim 1, wherein said rooting induction medium is 1/2MS +0.5mg/LIBA medium.
8. The tissue culture method of morinda citrifolia of claim 1, wherein said explants comprise young shoot segments, semi-lignified shoot segments, and fully lignified shoot segments.
9. The tissue culture method of morinda citrifolia of claim 1, further comprising explant conditioning, said explant conditioning comprising: transplanting morinda officinalis plants to a greenhouse, removing the rattan, spraying carbendazim solution at intervals, and taking new rattan as the explant after the new rattan grows out.
10. The tissue culture method of morinda officinalis how of claim 9, wherein the concentration of said carbendazim solution is 8% -12% by mass, and said carbendazim solution is sprayed every 5-8 days.
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| CN115024221A (en) * | 2022-06-15 | 2022-09-09 | 广州中医药大学(广州中医药研究院) | Method for rapidly propagating tissue culture seedlings of large-leaf morinda officinalis and application of method |
| CN116965336A (en) * | 2023-09-13 | 2023-10-31 | 广州中医药大学(广州中医药研究院) | Method for rapid propagation of wild morinda officinalis tissue culture |
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| CN116965336A (en) * | 2023-09-13 | 2023-10-31 | 广州中医药大学(广州中医药研究院) | Method for rapid propagation of wild morinda officinalis tissue culture |
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