CN112544444B - Tissue culture medium for manglietia insignis, method for culturing embryonic callus of manglietia insignis and method for rapidly propagating manglietia insignis - Google Patents
Tissue culture medium for manglietia insignis, method for culturing embryonic callus of manglietia insignis and method for rapidly propagating manglietia insignis Download PDFInfo
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Images
Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G31/00—Soilless cultivation, e.g. hydroponics
-
- 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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- Life Sciences & Earth Sciences (AREA)
- Developmental Biology & Embryology (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Environmental Sciences (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention relates to the field of plant cultivation, in particular to a tissue culture medium for manglietia insignis, a method for cultivating embryonic callus of manglietia insignis and a method for rapidly propagating manglietia insignis. The invention provides a tissue culture medium for manglietia insignis, which comprises an induction medium; the induction culture medium takes a WPM culture medium as a basic culture medium, and also comprises the following components in percentage by weight: 2, 4-D1-4 mg/L, 6-BA 0-1 mg/L, PVP 1-3 g/L, CH 0.5-2 g/L, sucrose 20-50 g/L and plant gel 3-4 g/L; the pH value of the culture medium for induction culture is 4-7. The manglietia insignis cultured by the culture medium provided by the invention has high rooting rate, and can realize rapid and mass propagation of manglietia insignis plants.
Description
Technical Field
The invention relates to the field of plant cultivation, in particular to a tissue culture medium for manglietia insignis, a method for cultivating embryonic callus of manglietia insignis and a method for rapidly propagating manglietia insignis.
Background
Manglietia sativa (Wall.) Bl.) is an evergreen broad-leaved arbor of the genus Mangliaceae (Magnolia) of the family Magnoliaceae (Magnoliaceae). The tree shape is beautiful, the leaf shape is beautiful, the flower is large and gorgeous and red, the flower has fragrance and certain anti-fouling effect, and the tree is an excellent tree species for garden landscape and urban greening. The wood has straight grains and easy processing, and is also an excellent decorative material and a plywood tree species. The plants are scattered, small in quantity and continuously cut, so that the plants meet the endangered dead places at present and are classified as national third-level protection plants.
At present, tender stem sections, plant leaves, petioles, terminal buds, lateral buds and the like of seeds are mostly adopted for tissue culture of the manglietia insignis. These explants are difficult to disinfect and browning is difficult to control; moreover, although these explants can successfully induce callus or regenerated buds, the explants have the problems of difficult rooting, low efficiency and the like.
Disclosure of Invention
In view of the above, the invention provides a tissue culture medium for manglietia insignis, a method for culturing embryonic callus of manglietia insignis and a method for rapidly propagating manglietia insignis.
In order to achieve the above purpose, the invention provides the following technical scheme:
the invention provides a tissue culture medium for manglietia insignis, which comprises an induction medium; the induction culture medium takes a WPM culture medium as a basic culture medium, and also comprises the following components in percentage by weight: 2, 4-D1-4 mg/L, 6-BA 0-1 mg/L, PVP 1-3 g/L, CH 0.5-2 g/L, sucrose 20-50 g/L and plant gel 3-4 g/L;
the pH value of the culture medium for induction culture is 4-7.
Preferably, the tissue culture medium further comprises a purification medium, a subculture multiplication medium and a differentiation medium;
the purification culture medium takes a WPM culture medium as a basic culture medium and also comprises the following components: 2, 4-D1-4 mg/L, 6-BA 0-0.5 mg/L, active carbon 0.5-3 g/L, PVP 0-3 g/L, CH 0.5-2 g/L, sucrose 20-50 g/L and plant gel 3-4 g/L;
the subculture multiplication medium takes a WPM medium as a basic medium and also comprises the following components: 1-4 mg/L of 2,4-D, 0.5-3 g/L, PVP 0-3 g/L, CH 0.5.5-2 g/L of active carbon, 20-50 g/L of cane sugar and 3-4 g/L of plant gel;
the differentiation medium takes a WPM medium as a basic medium and also comprises the following components: 0.5-3 g/L of active carbon, 20-50 g/L of cane sugar and 3-4 g/L of plant gel;
the pH value of the purification culture medium, the secondary multiplication culture medium and the differentiation culture medium is 4-7.
The invention provides application of the tissue culture medium in the technical scheme in the culture of the embryonic callus of the manglietia insignis.
The invention provides a method for culturing embryonic callus of manglietia insignis, which comprises the following steps:
(1) taking immature manglietia insignis fruits as explants;
(2) sterilizing the explant to obtain a sterilized fruit;
(3) cutting the disinfected fruit, and stripping immature seeds;
(4) dividing the immature seed into two halves, and performing induction culture with a downward section to obtain embryogenic callus;
(5) performing purification culture and subculture proliferation culture on the embryonic callus to obtain an embryonic cell line;
(6) carrying out differentiation culture on the embryonic cell mass in the embryonic cell line to obtain a somatic embryo;
(7) and (3) performing germination culture and tissue culture on the somatic embryos to obtain tissue culture seedlings.
Preferably, the immature fruits in the step (1) are fruits 3-5 weeks after the rosebush petals are completely opened.
Preferably, the induction culture in the step (4) is dark culture, the temperature is constant at 22-28 ℃, and the humidity is 40-60%.
Preferably, the purification culture and the subculture proliferation culture in the step (5) are performed in a dark culture mode, the temperature is 22-28 ℃, and the humidity is 40-60%.
Preferably, the differential culture mode is dark culture, the temperature is 22-28 ℃, the time is 14-42 days, and the humidity is 40-60%;
the temperature of germination culture is 22-28 ℃, the photoperiod is 12-16 h/8-12 h, the humidity is 40-60%, and the illumination intensity is 50-80 mu mol m-2s-1。
The invention provides a method for rapidly propagating manglietia insignis, which comprises the following steps: adopting the culture medium of the technical scheme to culture the obtained tissue culture seedling or adopting the method of the technical scheme to culture the embryonic callus of the manglietia insignis to obtain the tissue culture seedling; and (4) transplanting and culturing the tissue culture seedlings to obtain the manglietia insignis seedlings.
Preferably, the culture medium used in the transplanting comprises humus soil and turf; the volume ratio of the humus soil to the turf in the culture medium is 1: 1-3.
The invention provides a tissue culture medium for manglietia insignis, which comprises an induction medium; the induction culture medium takes a WPM culture medium as a basic culture medium, and also comprises the following components in percentage by weight: 2, 4-D1-4 mg/L, 6-BA 0-1 mg/L, PVP 1-3 g/L, CH 0.5-2 g/L, sucrose 20-50 g/L and plant gel 3-4 g/L; the pH value of the culture medium for induction culture is 4-7. In the combined culture medium provided by the invention, 2,4-D and 6-BA are plant growth regulating hormones for inducing embryogenic callus, PVP inhibits browning in the induction process, CH regulates plant osmotic pressure and provides nutrients for plants, sucrose provides a carbon source for plants, and plant gel solidifies the culture solution; the invention solves the problems of difficult rooting and low efficiency of the manglietia insignis through the matching of all components and the use amount and relative proportion of all the components.
The invention provides a method for culturing embryonic callus of manglietia insignis, which takes immature fruits of manglietia insignis as explants; sterilizing the explant to obtain a sterilized fruit; cutting the disinfected fruit, and stripping immature seeds; dividing the immature seed into two halves, and performing induction culture with a downward section to obtain embryogenic callus; culturing the embryonic cell mass to obtain an embryonic cell line; and culturing the embryonic cell mass in the embryonic cell line to obtain the tissue culture seedling. According to the invention, immature seeds are selected as explants, so that the explants are easier to disinfect successfully, the pollution rate is low, the generated browning substances are less, and the browning is controllable, thereby solving the problems of difficult disinfection and browning in the prior art and ensuring the rooting rate and efficiency of plants; the invention obtains the tissue culture seedling by the culture of the embryogenesis way (namely the process that immature seeds of the manglietia insignis undergo induction culture to generate embryogenic callus), solves the problem that the tissue culture way and the asexual propagation way need to perform rooting culture on the explant to obtain a complete plant, and avoids the condition of low efficiency caused by low rooting rate and serious browning of the rooting part. The method provided by the invention cultures the embryonic cell mass to obtain a tissue culture seedling; compared with other tissue culture methods, the method does not need rooting culture (namely the somatic embryo of the manglietia insignis and the zygotic embryo of the seed in the manglietia insignis have the capacity of directly developing into a finished plant, and the root can be differentiated by itself); and moreover, one zygote can only form one zygote embryo under the natural condition of the manglietia insignis, but one embryogenic callus can form a plurality of somatic embryos, so that the propagation efficiency of the manglietia insignis is improved. The technical scheme provided by the invention has the technical characteristics of high rooting rate, high efficiency, high speed, high seedling rate and high technical practicability.
In addition, the method provided by the invention is not influenced by environmental factors, the somatic embryo can be directly generated from the embryonic tissue of the manglietia insignis, the processes of blooming, fertilization and fertilized egg development into mature zygotic embryo and seed formation of normal seeds are skipped, the seedling period is shortened, the propagation is not influenced by climatic conditions, and the propagation and differentiation can be carried out for a long time.
The method provided by the invention has high efficiency of converting the embryonic callus into the somatic embryo and converting the somatic embryo into the plant, and can be used for large-scale and batch production of the manglietia insignis tissue culture seedlings. Compared with the tissue culture technology of the manglietia insignis reported in the prior art, the tissue culture technology of the manglietia insignis has the technical characteristics of high efficiency, high speed, high seedling rate and high technical practicability.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required to be used in the embodiments will be briefly described below.
FIG. 1 shows the process of obtaining an explant of Manglietia crocata, wherein a is the fruit of Manglietia crocata; b is the position of cutting the manglietia insignis seeds in the fruit; c is the immature seed of the rosewood lotus;
FIG. 2 shows the induction of the embryonic cell line of Manglietia crocea, wherein a is the immature seed of Manglietia crocea; b is the manglietia insignis seeds which are evenly cut into two halves; c is the newly induced embryonic callus of manglietia insignis;
FIG. 3 shows the process of the purification and differentiation of the embryonic cell line of Manglietia crocea, wherein a is the newly induced embryonic callus of Manglietia crocea; b is purified embryonic callus of manglietia insignis; c is the somatic embryo of the embryonic callus differentiation of the manglietia insignis; d, e is somatic embryo of manglietia insignis; f is the plant of the manglietia insignis of the somatic embryo development;
FIG. 4 shows the transplantation and domestication of the tissue culture seedlings of Manglietia rubra, wherein a is somatic cell differentiation seedling of Manglietia rubra; b is the tissue culture bottle seedling of the manglietia insignis; c is a 32-hole plug seedling of the manglietia insignis; d is a 16 multiplied by 16cm bag seedling of manglietia haematocephala.
Detailed Description
The invention provides a tissue culture medium for manglietia insignis, which comprises an induction medium; the induction culture medium takes a WPM culture medium (woody plant culture medium) as a basic culture medium, and also comprises the following components in percentage by weight: 2, 4-D1-4 mg/L, 6-BA 0-1 mg/L, PVP 1-3 g/L, CH 0.5-2 g/L, sucrose 20-50 g/L and plant gel 3-4 g/L; more preferably comprises the following content components: 2, 4-D2 mg/L, 6-BA 0.25mg/L, PVP 1g/L, CH 1g/L, sucrose 40g/L and plant gel 3 g/L; the pH value of the culture medium for induction culture is 4-7, and more preferably 5.7-5.8; in the present invention, the WPM medium preferably includes the following components: NH (NH)4N03 400mg/L、Ca(NO3)2·4H2O 556mg/L、K2SO4 990mg/L、CaCl2·2H2O 96mg/L、KH2PO4 170mg/L、H3BO3 6.2mg/L、 Na2MoO4·2H2O 0.25mg/L、MgSO4·7H2O 370mg/L、MnSO4·H2O 22.4mg/L、 ZnSO4·7H2O 8.6mg/L、CuSO4·5H2O 0.25mg/L、FeSO4·7H2O 27.8mg/L、 Na237.3mg/L EDTA, 100mg/L inositol, 11.0 mg/L vitamin B, 0.5mg/L nicotinic acid, 60.5 mg/L vitamin B, 2.0mg/L glycine, 30g/L sucrose and 3g/L plant gel; the WPM medium preferably has a pH of 5.7. In the present invention, the 2,4-D is preferably 2, 4-Dichlorophenoxyacrylic acid; the 6-BA is preferably N-6-Benzyladenine; the PVP is preferably polyvinylpyrrolindone; the CH is preferably Casein hydrosate; the plant gel is preferably Phytagel. In the combined culture medium provided by the invention, 2,4-D and 6-BA are plant growth regulating hormones for inducing embryogenic callus, and PVP inhibits browning in the induction processThe CH regulates the osmotic pressure of the plant and provides nutrients to the plant, sucrose provides a carbon source for the plant, and the plant gel solidifies the culture.
In the present invention, the tissue culture medium preferably further comprises a purification medium, a subculture multiplication medium and a differentiation medium;
the purification culture medium preferably takes a WPM culture medium as a basic culture medium, and preferably further comprises the following components: 2, 4-D1-4 mg/L, 6-BA 0-0.5 mg/L, active carbon 0.5-3 g/L, PVP 0-3 g/L, CH 0.5-2 g/L, sucrose 20-50 g/L and plant gel 3-4 g/L; more preferably comprises the following components: 2, 4-D2 mg/L, 0.25mg/L of 6-BA, 1g/L, PVP 1g/L, CH 1g/L of activated carbon, 40g/L of cane sugar and 3g/L of plant gel; the pH value of the purification culture medium is 4-7, and more preferably 5.7-5.8; the subculture multiplication medium preferably takes a WPM medium as a basic medium, and preferably further comprises the following components: 1-4 mg/L of 2,4-D, 0.5-3 g/L, PVP 0-3 g/L, CH 0.5.5-2 g/L of active carbon, 20-50 g/L of cane sugar and 3-4 g/L of plant gel; more preferably comprises the following components: 2, 4-D1 mg/L, activated carbon 1g/L, PVP 1g/L, CH 1g/L, sucrose 30g/L and plant gel 3 g/L; the pH value of the subculture multiplication medium is 4-7, and more preferably 5.7-5.8; the differentiation medium preferably takes a WPM medium as a basic medium, and preferably further comprises the following components: 0.5-3 g/L of active carbon, 20-50 g/L of cane sugar and 3-4 g/L of plant gel; more preferably comprises 1g/L of active carbon, 30g/L of cane sugar and 3g/L of plant gel; the pH value of the differentiation medium is 4-7, and more preferably 5.7-5.8.
The invention provides application of the tissue culture medium in the technical scheme in the culture of the embryonic callus of the manglietia insignis.
The invention provides a method for culturing embryonic callus of manglietia insignis, which comprises the following steps:
(1) taking immature manglietia insignis fruits as explants;
(2) sterilizing the explant to obtain a sterilized fruit;
(3) cutting the disinfected fruit, and stripping immature seeds;
(4) dividing the immature seed into two halves, and performing induction culture with a downward section to obtain embryogenic callus;
(5) performing purification culture and subculture proliferation culture on the embryonic callus to obtain an embryonic cell line;
(6) culturing the embryonic cell mass in the embryonic cell line to obtain a somatic embryo;
(7) and (3) performing germination culture and tissue culture on the somatic embryos to obtain tissue culture seedlings.
The invention takes the immature fruit of rosewood lotus as an explant. In the invention, the immature fruit is preferably a fruit 3-5 weeks after the rosewood lotus petals are completely opened. According to the invention, immature fruits are selected, and the induction rate of inducing somatic embryogenesis is higher than that of mature seeds; the material is easy to disinfect, and the pollution rate is low; and the material is fresh and tender, and the superclean bench is beneficial to cutting.
The invention carries out disinfection treatment on the explant to obtain the disinfected fruit. In the present invention, the sterilization treatment preferably comprises washing the explant; the cleaning mode is preferably that a brush stained with a detergent is used for cleaning under tap water; the detergent is preferably a conventional detergent or tween 20; the invention does not require any particular detergent, and detergents well known to those skilled in the art can be used. In the present invention, the sterilization treatment preferably includes a first sterilization treatment, a second sterilization treatment, and a third sterilization treatment; the first treatment is preferably carried out by immersing the fruit in NaDCC (Sodium Dichloroisocyanurate solution); the mass concentration of NaDCC is preferably 1-5 g/L, and more preferably 1 g/L; the soaking time is preferably 1-4 h, and more preferably 2 h; the second treatment mode is preferably that the fruits after the first disinfection are soaked in alcohol; the percentage content of the alcohol by volume is preferably 70-75%, and more preferably 75%; the soaking time is preferably 0-10 min, and more preferably 5 min; the third treatment mode is preferably that the fruits after the second disinfection are placed in alcohol to be soaked in NaDCC solution; the mass concentration of NaDCC is preferably 3-6 g/L, and more preferably 5 g/L; the soaking time is preferably 5-30 min, and more preferably 20 min. The invention preferably uses sterile water to wash the third sterilized fruit; the washing frequency of the sterile water is preferably 1-5 times, and more preferably 3 times. According to the invention, the sterilization mode can be adopted to achieve the purposes of more sufficient sterilization, reduction of explant pollution rate, small damage to explants and improvement of the survival rate of materials.
The invention provides a method for obtaining disinfected fruits, which comprises the steps of cutting the disinfected fruits and stripping immature seeds. In the present invention, the incising tool is preferably a forceps or a scalpel; the incision is preferably performed under a dissecting scope.
The invention divides the immature seed into two halves, and the section is downwards induced and cultured to obtain the embryogenic callus. In the present invention, the induction medium used in the induction culture is described in the above technical scheme, and is not described herein again. In the present invention, the induction culture is preferably performed in a dark culture; the temperature of the induction culture is preferably constant at 22-28 ℃, and more preferably 25 +/-2 ℃; the humidity of the induction culture is preferably 40-60%, and more preferably 40-50%. In the present invention, the induction culture is preferably performed in a closed culture dish; the culture dish is preferably a culture dish with the diameter of 9 cm; the dosage of the induction culture medium is preferably 20-30 ml, and more preferably 25 ml; the humidity for induction culture is preferably the humidity in a sealed culture dish when the culture dish is placed outside for induction culture. The invention preferably transfers explants to a new culture medium for induction culture every 14-42 days until embryogenic callus is induced, more preferably 28 days.
After obtaining the embryonic callus, the invention carries out purification culture and subculture proliferation culture on the embryonic cell mass to obtain an embryonic cell line. In the present invention, the purification medium and the subculture medium used in the purification culture and the subculture are described above and will not be described herein. In the present invention, the purification culture and the subculture proliferation culture are preferably performed in a closed culture dish; the culture dish is preferably a culture dish with the diameter of 9 cm; the dosage of the purification culture medium and the dosage of the subculture multiplication culture medium are preferably 20-30 ml respectively, and more preferably 25ml respectively; the humidity of the purification culture and the humidity of the subculture are preferably the humidity in a sealed culture dish when the culture dish is placed for external purification culture and subculture. In the present invention, the embryogenic cell mass is preferably a well-grown, undifferentiated, non-browned embryogenic cell mass; the purification culture mode is preferably dark culture; the temperature of the purification culture is preferably 22-28 ℃, and more preferably 25 +/-2 ℃; the humidity of the purification culture is preferably 40-60%, and more preferably 40-50%; the period of purification is preferably 1-3 weeks, and more preferably 2 weeks; the mode of the subculture is preferably dark culture; the temperature of the subculture is preferably 22-28 ℃, and more preferably 25 ℃; the humidity of the subculture is 40-60%, and more preferably 40-50%; the mode of the subculture is preferably dark culture; the temperature of the subculture is preferably constant at 22-28 ℃, and more preferably 25 +/-2 ℃; the humidity of the subculture is preferably 40-60%, and more preferably 40-50%. The subculture is preferably subcultured for 1 time every 1-4 weeks, and more preferably for 2 weeks; in the subculture, it is preferable to select an embryogenic cell mass that grows well, is undifferentiated, and is browned. The invention can obtain the embryonic cell line with vigorous growth and good state by 3 rounds of purification, culture and selection. And performing proliferation culture on the purified embryogenic callus on a subculture medium. The embryonic cell line can be proliferated by about 1 time in two weeks, and a large amount of embryonic cell lines can be obtained by subculture. The setting of the specific flowering culture and multiplication culture conditions can obtain embryonic cells with strong reproductive capacity and consistent growth state, and the purification culture improves the quality of the embryonic callus and is beneficial to multiplication and differentiation of the embryonic callus to somatic embryos.
After obtaining the embryonic cell line, the invention carries out differentiation culture on the embryonic cell mass in the embryonic cell line to obtain a somatic embryo. In the present invention, the differentiation medium used in the differentiation culture is described above and will not be described herein. In the invention, the differentiation culture method is preferably to culture the embryonic cell mass obtained by subculture in a differentiation medium to obtain a somatic embryo; during the differentiation culture, cell clusters obtained by subculture for 1-4 weeks are preferably selected, and more preferably for 2 weeks; the differentiation culture mode is preferably dark culture; the temperature of the differentiation culture is preferably constant at 22-28 ℃, and more preferably 25 +/-2 ℃; the humidity of the differentiation culture is preferably 40-60%, and more preferably 40-50%. In the present invention, the differentiation culture is preferably performed in a closed culture dish; the culture dish is preferably a culture dish with the diameter of 9 cm; the dosage of the differentiation culture medium is preferably 20-30 ml, and more preferably 25 ml; the humidity of the differentiation culture is preferably the humidity in a closed culture dish when the culture dish is placed outside for the differentiation culture. After 1 month of culture, the embryonic cell mass can differentiate a large number of somatic embryos. The invention can rapidly and efficiently propagate the tissue culture seedling of the manglietia insignis through a somatic cell embryo differentiation culture way. The method transfers the embryonic callus of the manglietia insignis to a culture medium without adding plant growth regulating hormone, so that the embryonic cells can be differentiated into somatic embryos by self in a large amount without expanding propagation, the differentiation speed of the embryonic callus is high, and 5-10 individual cell embryos can be differentiated from 0.1 g/group of the embryonic callus.
The somatic embryo is subjected to germination culture and tissue culture, and then tissue culture seedlings are obtained. In the present invention, the germination medium and the tissue culture medium used in the germination culture and the tissue culture have been described above and are not described herein. In the invention, the germination culture method is preferably to place the somatic embryo in a germination culture medium for culture to obtain a germinated somatic embryo; preferably, mature somatic embryos at the cotyledon embryo stage are selected for the germination culture; the selection is preferably carried out under a stereoscopic microscope; the germination culture medium used in the germination culture preferably adopts a differentiation culture medium; in the present invention, the differentiation medium has been described above and will not be described herein. In the invention, the temperature of germination culture is preferably constant at 22-28 ℃, and more preferably at 25 +/-2 ℃; the photoperiod of the germination culture is preferably 12-16 h/8-12 h, namely the illumination time of 12-16 h and the dark time of 8-12 h, and more preferably 16/8 h; the illumination intensity of the germination culture is preferably 50-80 mu mol m-2s-1(ii) a The sproutThe humidity of the fermentation culture is preferably 40-60%, and more preferably 50-60%. In the present invention, the germination culture is preferably performed in a closed petri dish; the culture dish is preferably a culture dish with the diameter of 9 cm; the dosage of the germination culture medium is preferably 20-30 ml, and more preferably 25 ml; the humidity for germination culture is preferably the humidity in a sealed culture dish when the culture dish is placed outside for germination culture. In the invention, the tissue culture method is preferably to place the germinated somatic embryos in a tissue culture bottle filled with WPM culture medium for continuous culture to obtain tissue culture seedlings. In the present invention, the tissue culture preferably selects somatic embryos in which somatic cells are just in the state where radicles are protruding and the daughter is also unfolded; the using amount of the WPM culture medium is preferably 30-100 ml, and more preferably 50 ml; the specification of the tissue culture bottle is preferably 250 ml; the WPM medium preferably comprises the following components: NH (NH)4N03 400mg/L、 Ca(NO3)2·4H2O 556mg/L、K2SO4 990 mg/L、CaCl2·2H2O 96mg/L、KH2PO4 170mg/L、H3BO36.2mg/L、Na2MoO4·2H2O 0.25mg/L、MgSO4·7H2O 370mg/L、 MnSO4·H2O 22.4mg/L、ZnSO4·7H2O 8.6mg/L、CuSO4·5H2O 0.25mg/L、 FeSO4·7H2O 27.8mg/L、Na237.3mg/L EDTA, 100mg/L inositol, 11.0 mg/L vitamin B, 0.5mg/L nicotinic acid, 60.5 mg/L vitamin B, 2.0mg/L glycine, 30g/L sucrose and 3g/L plant gel; the WPM medium preferably has a pH of 5.8; the tissue culture mode is preferably illumination culture; the temperature of the tissue culture is preferably 22-28 ℃, and more preferably 25 +/-2 ℃; the humidity of the tissue culture is preferably 40-60%, and more preferably 50-60%.
The invention also provides a rapid propagation method of the manglietia insignis, which comprises the following steps: obtaining a tissue culture seedling by adopting the method for culturing the embryonic callus of the manglietia insignis in the technical scheme; and (4) transplanting and culturing the tissue culture seedlings to obtain the manglietia insignis seedlings. In the invention, before the transplanting culture, the plant gel on the tissue culture seedling is preferably cleaned in tap water, and the selection standard of the tissue culture seedling is preferably that the length of the root is more than 5cm, and the number of fibrous roots is more than 2. In the invention, the culture medium used in the transplanting comprises humus soil and turf; the volume ratio of humus soil to turf in the culture medium is 1: 1-3; the sterilization mode of the culture substrate is preferably high-temperature moist heat sterilization; the sterilization is preferably carried out in a sterilization pot; the temperature of the sterilization is preferably 121 ℃; the time for sterilization is preferably 15-25 min, and more preferably 20 min. In the present invention, the transplanting culture preferably includes a nursery tray culture and a nursery bag culture; the mode of the seedling raising tray is preferably that the washed tissue culture seedlings are placed in the seedling raising tray filled with the culture medium and covered with a cover for culture, so as to obtain domesticated seedlings; the specification of the seedling raising plate is preferably 32 holes of 80mm multiplied by 560 mm; the seedling culture plate culture is preferably carried out in a glass greenhouse; the temperature of the seedling raising plate is preferably 20-30 ℃, and more preferably 25 +/-2 ℃; the light period of the seedling raising plate is preferably natural illumination; the time for culturing the seedling raising plate is preferably 2-5 weeks, and more preferably 3 weeks; in the invention, the seedling bag culture is preferably to transfer domesticated seedlings with more than 3 new leaves into a seedling bag filled with a culture medium for culture, and more preferably 4 seedlings; the specification of the seedling raising bag is preferably 16 x 16 cm; the seedling bag cultivation is preferably carried out in a glass greenhouse; the temperature of the seedling raising bag is preferably 20-28 ℃, and more preferably 25 +/-2 ℃; the light period of the seedling raising bag culture is preferably natural illumination; the time for culturing the seedling raising bag is preferably 2-4 months, and more preferably 3 months. The transplanting survival rate of the tissue culture seedlings of the manglietia insignis can be improved by adopting the transplanting culture provided by the invention, and the survival rate can reach more than 90%.
For further illustration of the present invention, the tissue culture medium for manglietia insignis, the method for culturing embryonic callus of manglietia insignis, and the method for rapid propagation of manglietia insignis provided by the present invention are described in detail below with reference to the accompanying drawings and examples, but they should not be construed as determining the scope of the present invention.
Example 1
Surface disinfection of manglietia insignis and manglietia insignis fruits
(1) Collecting immature fruits of the rosewood lotus petals about 3-5 weeks after the rosewood lotus petals are completely opened from the rosewood lotus plants.
(2) The brush is stained with detergent and washed clean under tap water. The fruit was then soaked in 1g/L Sodium Dichloroisocyanurate solution (NaDCC) for 2 hours. After the treatment, the seeds are transferred into a clean bench, sterilized in 75% alcohol for 5 minutes, treated in 5g/L NaDCC solution for 20 minutes, and washed with sterile water for 3 times.
(3) After the surface sterilization is finished, under a dissecting mirror, the fruit is cut open by using forceps and a scalpel to strip the manglietia insignis seeds, and the process for obtaining the manglietia insignis explants is shown in figure 1.
Secondly, inducing embryogenic callus by immature seeds
(4) Under a body type microscope, the obtained manglietia insignis seeds are evenly divided into two halves by a scalpel, and the section is placed on an induction culture part downwards. The formula of the induction culture medium is as follows: woody Plant Medium (WPM) +2 mg/L2, 4-dichlorphenoxygenic acid (2,4-D) +0.25mg/L N-6-Benzyladenine (6-BA) +1g/L Polyvinylpyrrolidone (PVP) +1g/L Casein Hydrosate (CH) +40g/L sucrose +3g/L Phytagel (plant gel), pH 5.7-5.8. The culture condition is constant temperature dark culture at 25 +/-2 ℃. The explants were transferred to new induction medium every 1 month until embryogenic callus of manglietia carthami was induced, and the induction process of the embryonic cell line of manglietia carthami is shown in fig. 2.
And thirdly, purifying and proliferating the embryonic callus of the manglietia insignis.
(5) The embryonic callus which is just induced has stronger differentiation capability, is usually mixed with the non-embryonic callus, and is easy to brown. Transferring the embryonic cell mass which grows well, is not differentiated and is not browned into a purification culture medium to culture under a stereoscopic microscope, purifying once every 2 weeks, selecting the embryonic cell mass which grows well, is not differentiated and is not browned during purification, and removing the browned cell mass and somatic embryos; the culture medium during the purification culture is as follows: woody plant culture medium (WPM) +2 mg/L2, 4-dichlorphenoxyacetic acid (2,4-D) +0.25mg/L N-6-benzyladine (6-BA) +1g/L active carbon +1g/L polyhydroxylated PVP) +1g/L Casein Hydrostate (CH) +40g/L sucrose +3g/L Phytagel, pH 5.7-5.8, wherein the induction medium is used in an amount of 25ml and the humidity is 40-60%. The embryonic cell line of the manglietia insignis which grows vigorously and is in a good state can be obtained through 3 rounds of purification, culture and selection. Transferring the manglietia insignis embryonic cell line to a subculture multiplication medium for propagation, performing subculture once every 2 weeks for 10 groups/dish, and performing multiplication on each dish of embryonic cells for two weeks to obtain 2 dishes. The subculture multiplication medium comprises: woody Plant Medium (WPM) +1 mg/L2, 4-dichlorphenoxygenic acid (2,4-D) +1g/L active carbon +1g/L polyvinylpyrrodine (PVP) +1g/L Casein Hydrostate (CH) +30g/L sucrose +3g/L Phytagel (plant gel), pH 5.7-5.8, the amount of the subculture medium in each culture dish is 25ml, the humidity is 40-60%, the purification and differentiation process of the mangrove embryonic cell line is shown in FIG. 3, FIG. 3 shows the whole differentiation, germination and seedling formation process, a large amount of somatic embryos can be generated by few somatic cells in the mode provided by the invention, and the somatic embryos can be converted into complete plants.
Fourthly, transplanting and domesticating the embryonic callus of the manglietia insignis to differentiate somatic embryos, differentiated plants of the somatic embryos and tissue culture seedlings.
(6) Transferring the embryonic cell mass of the manglietia insignis after 2 weeks of subculture to a differentiation medium for differentiation culture. The composition of the differentiation medium is WPM +1g/L activated carbon +30g/L cane sugar +3g/L Phytagel, the pH value is 5.7-5.8, the dosage of the differentiation medium in each culture dish is 25ml, the humidity is 40-60%, and the culture condition is dark culture at constant temperature of 25 +/-2 ℃. After 1 month of culture, the embryogenic cell mass can differentiate into a large number of somatic embryos.
Mature somatic embryos at the cotyledon embryo stage are picked up under a stereomicroscope and transferred to a culture dish filled with 20mL of differentiation medium for germination culture. The culture conditions are constant temperature of 25 +/-2 ℃, light cycle of 16/8h, humidity of 40-60% and illumination intensity of 50-80 mu mol m-2s-1. When somatic cell radicle is stretched out and cotyledon is developed and unfolded, 50ml of the seed-coat is transferred into the seed-coatAnd (3) continuously performing tissue culture in a 250ml tissue culture bottle of the WPM basic culture medium under the constant temperature of 25 +/-2 ℃ and the humidity of 40-60% to obtain tissue culture seedlings.
(7) Taking the tissue culture seedling of the manglietia insignis which takes roots (the length of the roots is more than 5cm, the number of fibrous roots is more than 2) out of the tissue culture bottle. And (4) cleaning the plant gel attached to the tissue culture seedlings by using 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 performing moist heat sterilization at the high temperature of 121 ℃ for 20min in a sterilization pot. The sterilized culture medium is filled into a 32-hole seedling raising tray with the specification of 80mm multiplied by 560 mm. Transplanting the tissue culture seedlings into a plug tray in a glass greenhouse, covering the plug tray, keeping the humidity at 25 +/-2 ℃, and removing the cover after culturing for 3 weeks until 4 new leaves grow. Transferring the domesticated seedlings of 4 new leaves into a seedling raising bag with the thickness of 16 multiplied by 16cm and filled with a culture medium, culturing in the open air at the temperature of 25 +/-2 ℃, and obtaining the roselle seedlings after 3 months. The transplantation and domestication of the tissue culture seedlings of the manglietia insignis are shown in fig. 4, and the transplantation and domestication of the tissue culture seedlings of the manglietia insignis can be successfully realized by adopting the mode provided by the invention, and the method has the advantages of draft of transplantation survival rate, no variation of seedlings formed by somatic cells and high seedling survival rate.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (7)
1. A method for culturing embryonic callus of manglietia insignis is characterized by comprising the following steps:
(1) taking immature manglietia insignis fruits as explants;
(2) sterilizing the explant to obtain a sterilized fruit;
(3) cutting the disinfected fruit, and stripping immature seeds;
(4) dividing the immature seed into two halves, and performing induction culture with a downward section to obtain embryogenic callus; during the induction culture, the induction culture medium adopted during the induction culture is a WPM culture medium, 2mg/L, 4-D, 0.25mg/L, 6-BA, 1g/LPVP, 1g/L, CH, 40g/L, sucrose and 3g/L Phytagel plant gel, and the pH value is 5.7-5.8;
(5) performing purification culture and subculture proliferation culture on the embryonic callus to obtain an embryonic cell line; the purification culture medium adopted in the purification culture is WPM +2 mg/L2, 4-D +0.25 mg/L6-BA +1g/L active carbon +1g/L PVP +1g/LCH +40g/L cane sugar +3g/L plant gel, and the pH value is 5.7-5.8;
the subculture multiplication medium adopted in the subculture multiplication culture is WPM +1 mg/L2, 4-D +1g/L active carbon +1g/LPVP +1g/L CH +30g/L sucrose +3g/L plant gel, and the pH value is 5.7-5.8;
(6) carrying out differentiation culture on the embryonic cell mass in the embryonic cell line to obtain a somatic embryo; the differentiation medium adopted during differentiation culture is WPM +1g/L active carbon +30g/L sucrose +3g/L Phytagel, and the pH value is 5.7-5.8;
(7) carrying out germination culture and tissue culture on the somatic embryos to obtain tissue culture seedlings; the germination culture is carried out by picking out mature somatic embryos at a cotyledon embryo stage under a stereomicroscope and transferring the mature somatic embryos to a culture dish filled with 20mL of a differentiation culture medium; the differentiation medium is the differentiation medium of the step (6).
2. The method according to claim 1, wherein the immature fruit in the step (1) is a fruit 3-5 weeks after the manglietia rubra petals are completely opened.
3. The method according to claim 1, wherein the induction culture in the step (4) is dark culture, the temperature is constant at 22-28 ℃, and the humidity is 40-60%.
4. The method according to claim 1, wherein the purification culture and the subculture proliferation culture in the step (5) are performed in a dark culture at a temperature of 22-28 ℃ and a humidity of 40-60%.
5. The method according to claim 1, wherein the differentiation culture is performed in a dark culture at 22-28 ℃ for 14-42 days and at a humidity of 40-60%;
the temperature of germination culture is 22-28 ℃, the photoperiod is 12-16 h/8-12 h, the humidity is 40-60%, and the illumination intensity is 50-80 mu mol m-2s-1。
6. A method for rapidly propagating Manglietia rubra is characterized by comprising the following steps: obtaining a tissue culture seedling by adopting the method for culturing the embryonic callus of the manglietia insignis as claimed in any one of claims 1 to 5; and (4) transplanting and culturing the tissue culture seedlings to obtain the manglietia insignis seedlings.
7. The method according to claim 6, wherein the cultivation substrate used in the transplanting comprises humus soil and peat; the volume ratio of the humus soil to the turf in the culture medium is 1: 1-3.
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