CN111557240B - Method for rapidly propagating embryonic cells of mangnolia officinalis - Google Patents

Method for rapidly propagating embryonic cells of mangnolia officinalis Download PDF

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CN111557240B
CN111557240B CN202010332531.6A CN202010332531A CN111557240B CN 111557240 B CN111557240 B CN 111557240B CN 202010332531 A CN202010332531 A CN 202010332531A CN 111557240 B CN111557240 B CN 111557240B
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culture medium
culture
magnolia officinalis
officinalis
magnolia
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CN111557240A (en
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贾艳霞
林亮
马俊超
郁步竹
徐倩
高彦粉
李唯奇
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Kunming Institute of Botany of CAS
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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
    • 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
    • A01H4/008—Methods for regeneration to complete plants

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Abstract

The invention provides a method for rapidly propagating embryonic cells of mangnolia officinalis, and belongs to the technical field of plant tissue culture. The method comprises the following steps: 1) sterilizing the surface of the magnolia officinalis seeds and stripping zygote embryos; 2) inducing the zygotic embryo to obtain cortex Magnolia officinalis embryonic callus; 3) differentiating the embryonic callus of the mangnolia officinalis to obtain a somatic embryo; 4) differentiating a somatic embryo of the magnolia officinalis into a plant; 5) domestication and transplantation of tissue culture seedlings of mangnolia officinalis. The invention can produce somatic embryos in large scale by inducing, proliferating and differentiating the embryonic callus of the magnolia officinalis, and the somatic embryos can be further developed into complete plants. The method has the advantages of high transplanting survival rate, high seedling rate and capability of efficiently and quickly propagating magnolia officinalis plants.

Description

Method for rapidly propagating embryonic cells of mangnolia officinalis
Technical Field
The invention relates to the technical field of plant tissue culture, in particular to a method for rapidly propagating embryonic cells of mangnolia officinalis.
Background
Magnolia officinalis (Magnolia officinalis of Magnolia rehd. etwils) is a plant of Magnolia (Magnolia) of the Magnoliaceae family (Magnolia). The bark, root bark, branch bark, flower and fruit of Magnolia officinalis can be used as the medicine. Cortex Magnoliae officinalis is a traditional Chinese medicine, and has been used for more than two thousand years in ChinaHistory of pharmaceutical use. The main active ingredients of cortex Magnolia officinalis include magnolol, honokiol, magnoline, volatile oil, and beta-eudesmol, wherein the main active ingredient is magnolol (C)18H18O2) With honokiol (C)18H18O2) Has antitumor, antibacterial, antiulcer, antidepressant, and anticarious effects. The magnolia officinalis has not only an important medicinal value but also a higher ornamental value. Because the medicinal value of the wild magnolia officinalis is excessively felled, the resources and the distribution are sharply reduced, and the wild magnolia officinalis is classified as a national secondary protection wild plant. The magnolia officinalis seeds have low propagation speed and difficult cuttage propagation. Tissue culture of the meristem approach of magnolia was generally unsuccessful due to the difficulty in explant sterilization, severe browning, difficulty in rooting, and the like.
At present, most of tissue culture of the magnolia officinalis adopts tender stem segments, stem segments and leaves of plants, petioles, terminal buds, lateral buds and the like of seeds. These explants, while successful in inducing callus or regenerating shoots, all suffer from low efficiency.
Disclosure of Invention
The invention aims to provide a method for rapidly propagating embryonic cells of mangnolia officinalis, which has the advantage of high propagation efficiency.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a method for rapidly propagating embryonic cells of mangnolia officinalis, which comprises the following steps:
1) disinfecting magnolia officinalis seeds, and stripping zygote embryos;
2) inoculating the zygotic embryo into an induction culture medium, and carrying out induction culture at the temperature of 20-25 ℃ under the dark condition until a faint yellow embryonic cell mass is induced;
3) transferring the faint yellow embryonic cell mass into a somatic embryo differentiation and germination culture medium, performing differentiation culture for 25-35 days at the temperature of 20-25 ℃ under the dark condition, and taking a mature somatic embryo at the cotyledon embryo stage;
4) transferring the mature somatic embryos in the stage of cotyledon embryos into a somatic embryo differentiation and germination culture medium, performing first germination culture at the temperature of 20-25 ℃, transferring the mature somatic embryos into a WPM (woody plant medium) basal culture medium when somatic embryo roots stretch out and cotyledon development develops and expands, and performing second germination culture to obtain rooted magnolia officinalis tissue culture seedlings;
5) transferring the rooted magnolia officinalis tissue culture seedlings into a culture medium, and culturing in a greenhouse until the magnolia officinalis tissue culture seedlings grow 4 new leaves to obtain domesticated seedlings;
6) and transferring the domesticated seedlings into a culture medium, and culturing in the open air to obtain magnolia officinalis plants.
Preferably, the sterilization in step 1) is performed by: soaking the mangnolia officinalis seeds in a sodium dichloroisocyanurate aqueous solution A for 2-24 h for first disinfection, soaking the seeds subjected to the first disinfection in an ethanol aqueous solution for 3-8 min for second disinfection, and soaking the seeds subjected to the second disinfection in a sodium dichloroisocyanurate aqueous solution B for 20-30 min for third disinfection to obtain seeds subjected to third disinfection.
Preferably, the concentration of the sodium dichloroisocyanurate in the sodium dichloroisocyanurate aqueous solution A is 0.5-1.5 g/L; the volume percentage content of the ethanol in the ethanol water solution is 75 percent; the concentration of the sodium dichloroisocyanurate in the sodium dichloroisocyanurate aqueous solution B is 4-6 g/L.
Preferably, the induction medium in step 2) uses WPM as a basal medium, and further comprises the following components in mass concentration: 2, 4-D1.5-2.5 mg/L, 6-BA 0.2-0.3 mg/L, polyvinylpyrrolidone 0.8-1.2 g/L, casein hydrolysate 0.8-1.2 g/L, sucrose 38-42 g/L and plant gel 2.5-3.5 g/L; the pH value of the induction culture medium is 5.6-6.0.
Preferably, before transferring the yellowish embryonic cell mass into a somatic embryo differentiation and germination culture medium in the step 3), transferring the yellowish embryonic cell mass into an induction culture medium for subculture, continuously selecting the yellowish embryonic cell mass from the subculture to obtain a magnolia cortex embryonic cell line, and then performing differentiation culture.
Preferably, the number of subcultures is 2-3.
Preferably, the somatic embryo differentiation and germination culture medium in step 3) or step 4) takes WPM as a basic medium, and further comprises the following components in mass concentration: 0.8-1.2 g/L of active carbon, 28-32 g/L of cane sugar and 2.5-3.5 g/L of plant gel; the pH value of the somatic embryo differentiation and germination culture medium is 5.6-6.0.
Preferably, the sunshine conditions of the first germination culture in the step 4) are 16h of illumination and 8h of darkness, and the illumination intensity is 55-65 mu mol-2s-1。
Preferably, the time for culturing the second germination in the step 4) is 50-70 d.
Preferably, the culture medium in the step 5) or the step 6) comprises the following components in parts by volume: 0.8-1.2 parts of humus soil and 0.8-1.2 parts of grass carbon.
Compared with the prior art, the invention has the following beneficial effects:
1. compared with the traditional seed seedling culture, the method shortens the seedling culture period, gets rid of the limitation of the propagation by climatic conditions, utilizes the somatic embryos of the mangnolia officinalis to propagate the mangnolia officinalis, and can quickly and efficiently obtain the propagated mangnolia officinalis plants.
2. The zygotic embryo is selected as the explant, and compared with the explants such as stem segments, terminal buds, petioles, leaves and the like reported at present, the explant is easier to successfully disinfect, has low pollution rate and is controllable in browning.
3. The invention breeds magnolia officinalis plants by an embryogenesis way, is different from a tissue culture way and an asexual propagation way, and obtains complete plants by rooting culture of explants. Generally, the efficiency is not high due to low rooting rate and serious browning of the rooted part. The somatic embryo differentiated from the embryogenic callus induced by the invention can directly develop into a complete magnolia officinalis plant without rooting culture.
4. The efficiency of transforming the embryogenic callus into the somatic embryo and transforming the somatic embryo into the plant is extremely high, and the large-scale and batch production of the magnolia officinalis tissue culture seedling can be carried out. Other tissue culture approaches of the magnolia officinalis reported at present have no case of successfully regenerating a complete plant. Compared with the tissue culture technology of the magnolia officinalis reported in the prior art, the tissue culture technology of the magnolia officinalis has the technical characteristics of high efficiency, high speed, high seedling rate and high technical practicability.
Drawings
FIG. 1 shows the process of Magnolia bark seed treatment and zygote embryo decortication, wherein a in FIG. 1 is Magnolia bark fruit; b in FIG. 1 is Magnolia bark seed with red testa; in FIG. 1, c is the magnolia bark seed with the testa removed; in FIG. 1, d is the position of the Magnolia zygotic embryo in the endosperm; in FIG. 1, e is the zygotic embryo of Magnolia officinalis;
FIG. 2 shows the induction and purification process of callus derived from magnolia bark embryos, wherein a in FIG. 2 is the magnolia zygote embryo; b in FIG. 2 is the newly induced embryonic callus of Magnolia officinalis; c in FIG. 2 is the purified embryogenic callus of Magnolia officinalis;
FIG. 3 shows the process of differentiation of somatic embryos from embryogenic callus of Magnolia officinalis, wherein a in FIG. 3 is the purified embryogenic callus; in FIG. 3, b is the differentiation of somatic embryos from embryogenic callus; FIG. 3 c is a somatic embryo; d in FIG. 3 is somatic embryo germination;
FIG. 4 is a process of transplanting and acclimating tissue culture seedlings of Magnolia officinalis, wherein a in FIG. 4 is a tissue culture seedling formed by somatic embryo development; in the figure 4, b and c are both 1 year after the tissue culture seedlings of the magnolia officinalis are transplanted and domesticated.
Detailed Description
The invention provides a method for rapidly propagating embryonic cells of mangnolia officinalis, which comprises the following steps:
1) disinfecting magnolia officinalis seeds, and stripping zygote embryos;
2) inoculating the zygotic embryo into an induction culture medium, and carrying out induction culture at the temperature of 20-25 ℃ under the dark condition until a faint yellow embryonic cell mass is induced;
3) transferring the faint yellow embryonic cell mass into a somatic embryo differentiation and germination culture medium, performing differentiation culture for 25-35 days at the temperature of 20-25 ℃ under the dark condition, and taking a mature somatic embryo at the cotyledon embryo stage;
4) transferring the mature somatic embryos in the stage of cotyledon embryos into a somatic embryo differentiation and germination culture medium, performing first germination culture at the temperature of 20-25 ℃, transferring the mature somatic embryos into a WPM (woody plant medium) basal culture medium when somatic embryo roots stretch out and cotyledon development develops and expands, and performing second germination culture to obtain rooted magnolia officinalis tissue culture seedlings;
5) transferring the rooted magnolia officinalis tissue culture seedlings into a culture medium, and culturing in a greenhouse until the magnolia officinalis tissue culture seedlings grow 4 new leaves to obtain domesticated seedlings;
6) and transferring the domesticated seedlings into a culture medium, and culturing in the open air to obtain magnolia officinalis plants.
The method firstly disinfects the seeds of the magnolia officinalis and strips the zygote embryo.
In the invention, the magnolia officinalis seeds are preferably collected from healthy magnolia officinalis plants; the time of the collection is preferably 9 months; the Magnolia bark seeds are preferably obtained from red ripe Magnolia bark fruits; the magnolia officinalis seeds are preferably magnolia officinalis seeds with red episperm removed; in the specific implementation process of the invention, the red mature mangnolia officinalis fruits are peeled by a knife and a pair of pliers to take out the seeds, the seeds are wrapped by gauze, and the seeds are placed on a stainless steel screen to rub off the red episperm.
In the invention, the Magnolia officinalis seeds are preferably wrapped in gauze for disinfection in the disinfection process, and a detergent is added into the gauze; the detergent is a household detergent, the dosage of the detergent is not particularly limited, and the detergent is added to wash off stains on the surfaces of seeds; the gauze is used for wrapping the seeds, so that the seeds can be protected, and the embryo is prevented from being damaged during rubbing; the disinfection is preferably carried out by the following method: soaking magnolia officinalis seeds in a sodium dichloroisocyanurate (NaDCC) aqueous solution A for 2-24 h for first disinfection, soaking the first disinfected seeds in an ethanol aqueous solution for 3-8 min for second disinfection, soaking the second disinfected seeds in a sodium dichloroisocyanurate aqueous solution B for 20-30 min for third disinfection to obtain third disinfected seeds; the concentration of the sodium dichloroisocyanurate in the sodium dichloroisocyanurate aqueous solution A is preferably 0.5-1.5 g/L, and more preferably 1 g/L; the volume percentage content of the ethanol in the ethanol aqueous solution is preferably 75%; the concentration of sodium dichloroisocyanurate in the sodium dichloroisocyanurate aqueous solution B is preferably 4-6 g/L, and more preferably 5 g/L; the first disinfection time is preferably 5-20 hours, and more preferably 10-15 hours; the time of the second disinfection is preferably 5 min; the time of the third sterilization is preferably 25 min; after the third disinfection, preferably, the method further comprises the step of washing the third disinfected seeds with clear water; the number of washing is preferably 3.
In the invention, the method for stripping zygotic embryo is not particularly limited, and the conventional method for stripping zygotic embryo in the field can be adopted. In the specific implementation process of the invention, the zygotic embryo is stripped by adopting the following method: under a stereoscopic microscope, cutting off the seed coat of the magnolia officinalis seed by using forceps and a scalpel; the top of the endosperm (the zygotic embryo is deep in the endosperm) is cut with a scalpel, and the zygotic embryo is cut with a scalpel.
Inoculating the zygotic embryo into an induction culture medium, and carrying out induction culture at the temperature of 20-25 ℃ under the dark condition until a faint yellow embryonic cell mass is induced.
The induction culture product of the invention comprises a browned cell mass and a somatic embryo which are removed besides a faint yellow embryonic cell mass. In the specific implementation process of the invention, a light yellow embryonic cell mass is selected, and a browned cell mass and a somatic embryo are removed. This is because the embryogenic callus which has just been induced has a strong differentiation ability, but is often mixed with non-embryogenic callus and is easily browned. Purified embryogenic callus is obtained by selecting embryogenic cell mass and propagation culture.
In the present invention, the selection of the yellowish embryogenic cell mass is performed under a microscope.
In the present invention, the induction medium uses WPM (woody plant medium) as a basic medium, and preferably further includes the following components by mass: 2,4-D (2, 4-dichlorphenoxyacetic acid) 1.5-2.5 mg/L, 6-BA (N-6-Benzyladenine) 0.2-0.3 mg/L, PVP (Polyvinylpyrrolidone ) 0.8-1.2 g/L, CH (Casein hydrolysate) 0.8-1.2 g/L, sucrose 38-42 g/L and plant gel 2.5-3.5 g/L, more preferably, the induction medium takes WPM as a basic medium and further comprises the following components in mass concentration: 2,4-D2mg/L, 6-BA0.25mg/L, PVP1g/L, CH1g/L, 40g/L of cane sugar and 2.5-3.5 g/L of plant gel; the pH value of the induction culture medium is preferably 5.6-6.0, and more preferably 5.8.
In the invention, in the induction culture process, the zygotic embryos (explants) are transferred to a new induction culture medium for continuous culture every 1 month.
After obtaining a faint yellow embryonic cell mass, transferring the faint yellow embryonic cell mass into a somatic embryo differentiation and germination culture medium, carrying out differentiation culture for 25-35 d at the temperature of 20-25 ℃ under a dark condition, and taking a mature somatic embryo at a cotyledon embryo stage; the time for the differentiation culture is preferably 30 d.
Before transferring the faint yellow embryonic cell mass into a somatic embryo differentiation and germination culture medium in the step 3), preferably, transferring the faint yellow embryonic cell mass into an induction culture medium for subculture, continuously selecting the faint yellow embryonic cell mass from a subculture to obtain a magnolia cortex embryonic cell line, and then performing differentiation culture; the number of times of subculture is preferably 2-3, and the time of each subculture is preferably 20-25 d; in the process of selecting the faint yellow embryonic cell mass, the browned cell mass and the somatic embryo are removed; the invention has the effect of subculture to obtain the magnolia officinalis embryonic cell line with vigorous growth and good state.
In the present invention, the somatic embryo differentiation and germination medium preferably uses WPM as a basal medium, and further comprises the following components by mass: 0.8-1.2 g/L of activated carbon, 28-32 g/L of sucrose and 2.5-3.5 g/L of plant gel, and preferably, the WPM is used as a basic culture medium, and the WPM further comprises the following components in mass concentration: 1g/L of activated carbon, 30g/L of cane sugar and 3g/L of plant gel; the pH value of the somatic embryo differentiation and germination culture medium is 5.6-6.0, and preferably 5.8.
After obtaining the mature somatic embryos in the cotyledon embryo stage, transferring the mature somatic embryos in the cotyledon embryo stage into a somatic embryo differentiation and germination culture medium, carrying out first germination culture at the temperature of 20-25 ℃, transferring into a WPM basal culture medium when somatic embryo roots stretch out and cotyledon development develops, and carrying out second germination culture to obtain rooted magnolia officinalis tissue culture seedlings.
In the present invention, the sunshine condition for the first germination culture or the second germination culture is independently preferably 16h of sunshineIn 8 hours of darkness and independent illumination intensity, the preferred illumination intensity is 55-65 mu mol-2s-1More preferably 60. mu. mol-2s-1(ii) a The time for the second germination culture is preferably 50-70 d, and more preferably 60 d.
After the rooted tissue culture seedling of the magnolia officinalis is obtained, the rooted tissue culture seedling of the magnolia officinalis is transferred into a culture medium and cultured in a greenhouse until the tissue culture seedling of the magnolia officinalis grows 4 new leaves, and domesticated seedlings are obtained.
In the present invention, the container for the culture is preferably a plug; the hole plate is preferably a seedling raising plate; the seedling raising plate is preferably a 32-hole seedling raising plate with the thickness of 80mm and 560 mm; the greenhouse is preferably a glass greenhouse; in the culture process, the cover of the plug tray is covered to keep humidity, the cover is removed after 3 weeks of culture, and the culture is continued.
In the present invention, the culture medium preferably comprises the following components in parts by volume: 0.8-1.2 parts of humus soil and 0.8-1.2 parts of grass carbon, and more preferably comprises the following components in parts by volume: 1 part of humus soil and 1 part of grass carbon. The culture substrate is preferably prepared by the following method: and mixing the humus soil and the turf, and sterilizing to obtain the culture medium.
After obtaining the domesticated seedling, transferring the domesticated seedling into a culture medium, and culturing in the open air to obtain a magnolia officinalis plant; the cultivation is preferably carried out in a nursery bag; the specification of the seedling raising bag is preferably 16 x 16 cm.
The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
1. Magnolia bark seed harvesting
In 9 months, red ripe magnolia fruits are collected from robust plants of magnolia officinalis. The fruit is peeled off with a knife and pliers to take out the seeds. Wrapping the seeds with gauze, and rubbing off the red testa on a stainless steel screen.
2. Surface disinfection of magnolia seeds
Wrapping cortex Magnolia officinalis with gauze, adding appropriate amount of detergent, weighing 0.1g NaDCC, diluting with 100mL distilled water to desired volume, transferring to 150mL glass triangular flask, and soaking for 2 hr. Transferring into a clean bench, transferring 60 seeds into a sterilized 250mL tissue culture bottle with forceps, and adding 50mL 75% (v/v) ethanol water solution for treatment for 5 min. Pouring off alcohol when the treatment is finished, adding 100mL of 5g/L NaDCC aqueous solution, and treating for 25min, wherein the triangular flask needs to be shaken during the treatment. At the end of the treatment, the NaDCC aqueous solution was decanted and washed three times with sterile water, 100mL each time with 3min of distilled water. After washing, the seeds were placed in a 9cm glass petri dish and the surface moisture of the seeds was blotted with sterile filter paper.
3. Peeling of magnolia officinalis zygotic embryo
The seed coat of Magnolia officinalis seed is cut with forceps and scalpel under stereomicroscope. The top of the endosperm was carefully dissected with a scalpel, which showed the zygotic embryo deep into the endosperm, and the zygotic embryo was dissected with a scalpel.
The processing of Magnolia officinalis seeds and the process of peeling zygotic embryos are shown in FIG. 1. Wherein a in figure 1 is magnolia officinalis fruit; b in FIG. 1 is Magnolia bark seed with red testa; in FIG. 1, c is the magnolia bark seed with the testa removed; in FIG. 1, d is the position of the Magnolia zygotic embryo in the endosperm; in FIG. 1, e is the zygotic embryo of Magnolia officinalis.
4. Zygotic embryo induced embryogenic callus
And (4) inoculating the stripped magnolia officinalis zygotic embryo on induction culture. The culture condition is constant temperature dark culture at 25 ℃. Transferring the explant to a new induction culture medium every 1 month until the callus of the magnolia officinalis embryo is induced.
The formula of the induction culture medium is as follows: woody Plant Medium (WPM) +2mg/L2, 4-dichlorphenoxycyanidic acid (2,4-D) +0.25mg/L N-6-benzyladine (6-BA) +1 g/lpolyvinylpyrrolidine (pvp) +1g/L Casein Hydrosate (CH) +40g/L sucrose +3g/LPhytagel (phytogel), ph 5.8.
5. Purification and proliferation of Magnolia officinalis embryogenic callus
The embryogenic callus which is just induced has strong differentiation capability, but is usually mixed with non-embryogenic callus and is easy to brown. Purified embryogenic callus is obtained by selecting embryogenic cell mass and propagation culture.
Selecting light yellow and undifferentiated embryogenic cell masses under a stereomicroscope, transferring the cells into an induction medium for subculture, subculturing for 1 time every 3 weeks, selecting the light yellow embryogenic cell masses during subculture, and removing browned cell masses and somatic embryos. Through 3 rounds of selection and enrichment culture, a magnolia embryonic cell line with vigorous growth and good state can be obtained.
The induction and purification process of Magnolia bark embryogenic callus is shown in FIG. 2. Wherein a in FIG. 2 is Magnolia officinalis zygotic embryo; b in FIG. 2 is the newly induced embryonic callus of Magnolia officinalis; c in FIG. 2 is the purified callus of Magnolia cortex.
6. The Magnolia officinalis embryonic callus is differentiated into somatic embryo, and the somatic embryo is differentiated into plant.
Transferring the magnolia bark embryonic cell mass after the subculture for 2 weeks into a somatic embryo differentiation and germination culture medium for differentiation culture. The culture condition is constant temperature dark culture at 25 ℃. After 1 month of culture, the embryogenic cell mass can differentiate into a large number of somatic embryos.
The composition of the somatic embryo differentiation and germination culture medium is WPM +1g/L active carbon +30g/L sucrose +3g/L Phytagel, and the pH value is 5.8.
The process of differentiation of somatic embryos from Magnolia embryogenic callus is shown in FIG. 3. Wherein a in FIG. 3 is the purified embryogenic callus; in FIG. 3, b is the differentiation of somatic embryos from embryogenic callus; FIG. 3 c is a somatic embryo; in FIG. 3 d is somatic embryo germination.
7. Formation of tissue culture seedling of Magnolia officinalis
When somatic cell radicle is stretched out and cotyledon develops and expands, transferring the somatic cell radicle into a 250mL tissue culture bottle filled with 50mLWPM basic culture medium for continuous culture. After 2 months of culture, the tissue culture seedling of the magnolia officinalis with good root system can be obtained.
8. A domestication and transplantation method of tissue culture seedlings of mangnolia officinalis.
Domestication and transplantation of tissue culture seedling of Magnolia officinalis, and taking out the rooted tissue culture seedling of Magnolia officinalis from tissue culture bottle. And (3) cleaning the plant gel attached to the tissue culture seedlings in tap water, and transferring the plant gel into a culture medium. The culture medium is humus soil and turf, and the ratio of the humus soil to the turf is 1: 1, and filling the sterilized culture medium into a 32-hole seedling raising tray with the specification of 80mm x 560 mm. In a glass greenhouse, the tissue culture seedlings are transplanted into a plug tray, a cover is covered to keep humidity, and the cover is removed after 3 weeks of culture until 4 new leaves grow. Transferring the domesticated seedlings of 4 new leaves into a seedling raising bag of 16 x 16cm filled with a culture medium, and culturing in the open air.
The transplanting and domesticating process of the magnolia officinalis tissue culture seedlings is shown in figure 4, wherein a in figure 4 is the tissue culture seedling formed by somatic embryo development; in the figure 4, b and c are both 1 year after the tissue culture seedlings of the magnolia officinalis are transplanted and domesticated.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art of the present invention, several modifications and improvements can be made without departing from the principle of the present invention, and these modifications and improvements should also be considered as the protection scope of the present invention.

Claims (6)

1. A method for rapidly propagating cortex magnoliae officinalis embryonic cells comprises the following steps:
1) disinfecting magnolia officinalis seeds, and stripping zygote embryos;
2) inoculating the zygotic embryo into an induction culture medium, and carrying out induction culture at the temperature of 20-25 ℃ under the dark condition until a faint yellow embryonic cell mass is induced;
3) transferring the faint yellow embryonic cell mass into a somatic embryo differentiation and germination culture medium, performing differentiation culture for 25-35 days at the temperature of 20-25 ℃ under the dark condition, and taking a mature somatic embryo at the cotyledon embryo stage;
4) transferring the mature somatic embryos in the stage of cotyledon embryos into a somatic embryo differentiation and germination culture medium, performing first germination culture at the temperature of 20-25 ℃, transferring the mature somatic embryos into a WPM (woody plant medium) basal culture medium when somatic embryo roots stretch out and cotyledon development develops and expands, and performing second germination culture to obtain rooted magnolia officinalis tissue culture seedlings;
5) transferring the rooted magnolia officinalis tissue culture seedlings into a culture medium, and culturing in a greenhouse until the magnolia officinalis tissue culture seedlings grow 4 new leaves to obtain domesticated seedlings;
6) transferring the domesticated seedlings into a culture medium, and culturing in the open air to obtain magnolia officinalis plants;
the induction culture medium in the step 2) consists of a basic culture medium, 2,4-D, 6-BA, polyvinylpyrrolidone, casein hydrolysate, sucrose and plant gel; the basic culture medium is WPM; the mass concentration of the 2,4-D is 1.5-2.5 mg/L, the mass concentration of the 6-BA is 0.2-0.3 mg/L, the mass concentration of the polyvinylpyrrolidone is 0.8-1.2 g/L, the mass concentration of the casein hydrolysate is 0.8-1.2 g/L, the mass concentration of the sucrose is 38-42 g/L, and the mass concentration of the plant gel is 2.5-3.5 g/L; the pH value of the induction culture medium is 5.6-6.0;
the somatic embryo differentiation and germination culture medium in the step 3) or the step 4) consists of a basic culture medium, activated carbon, cane sugar and plant gel; the basic culture medium is WPM; the mass concentration of the activated carbon is 0.8-1.2 g/L, the mass concentration of the sucrose is 28-32 g/L, and the mass concentration of the plant gel is 2.5-3.5 g/L; the pH value of the somatic embryo differentiation and germination culture medium is 5.6-6.0;
before transferring the faint yellow embryonic cell mass into a somatic embryo differentiation and germination culture medium in the step 3), transferring the faint yellow embryonic cell mass into an induction culture medium, carrying out subculture, continuously selecting the faint yellow embryonic cell mass from a subculture to obtain a magnolia officinalis embryonic cell line, and then carrying out differentiation culture.
2. The method according to claim 1, wherein the sterilization in step 1) is performed by: soaking magnolia officinalis seeds in a sodium dichloroisocyanurate aqueous solution A for 2-24 h for first disinfection, soaking the first disinfected seeds in an ethanol aqueous solution for 3-8 min for second disinfection, and soaking the second disinfected seeds in a sodium dichloroisocyanurate aqueous solution B for 20-30 min for third disinfection to obtain third disinfected seeds;
the concentration of sodium dichloroisocyanurate in the sodium dichloroisocyanurate aqueous solution A is 0.5-1.5 g/L; the volume percentage content of the ethanol in the ethanol water solution is 75 percent; the concentration of the sodium dichloroisocyanurate in the sodium dichloroisocyanurate aqueous solution B is 4-6 g/L.
3. The method according to claim 1, wherein the number of subcultures is 2 to 3.
4. The method according to claim 1, wherein the first germination culture in step 4) is performed under 16h illumination and 8h darkness with an illumination intensity of 55-65 μmol m-2s-1。
5. The method according to claim 1, wherein the second germination cultivation in step 4) is performed for 50-70 days.
6. The method as claimed in claim 1, wherein the cultivation substrate in step 5) or 6) consists of the following components in parts by volume: 0.8-1.2 parts of humus soil and 0.8-1.2 parts of grass carbon.
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