CN110663548A - Aseptic germination seedling raising method for platycerium wallichii spores - Google Patents
Aseptic germination seedling raising method for platycerium wallichii spores Download PDFInfo
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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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- 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
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Abstract
The invention provides a method for cultivating seedlings by aseptic germination of platycerium spores, which takes leaves with mature spores of platycerium as explant material-taking objects, designs a special culture medium formula for each culture stage, and achieves the purpose of rapid propagation through spores-GGB-buds, and comprises the following steps: selection and disinfection of explants, spore germination, GGB differentiation culture, rooting culture and test-tube plantlet transplantation. By adopting the method, only 178d to 210d can be used for obtaining the platycerium seedlings; the spore germination and the proliferation of GGB are synchronously performed, the culture link is simplified, the culture cost is reduced, the spore germination rate is high, the GGB proliferation rate is high, the seedling time is short, namely, the propagation efficiency is high, and the whole seedling culture efficiency is improved; in addition, the test-tube plantlets cultured by the method are strong and have developed root systems, and the environmental adaptability is strong through domestication and seedling hardening, so that the seedling transplanting survival rate is improved, and the purpose of rapid breeding of the platycerium seedlings is realized.
Description
[ technical field ] A method for producing a semiconductor device
The invention belongs to the technical field of plant tissue culture, and particularly relates to a sterile germination seedling raising method for platycerium spores.
[ background of the invention ]
Ceratopteris elaphus (Platycerium wallichii Hook.) is a perennial evergreen herbaceous plant of Ceratopteris of Ceratopteridae, and is native to dam of Yingjiang county in southwest of China, and has an altitude of 210 m in Yulin of 950 m mountain land, where Burma, eastern India, Thailand and China are also distributed. The species has been listed as the national secondary protection plant. The plant type is peculiar, the posture is beautiful, and the plant is a fern plant with the most peculiar posture in ornamental ferns. The leaves are divided into basal shield leaves and sporophyll, the basal shield leaves are also called sterile leaves or nutritive leaves, are round, oval or fan-shaped, and are attached to the rhizomes in a covering tile shape; the sporophyll is also called fertile leaf or normal leaf, is in a vertical or drooping shape, is mostly in a multi-branch shape, the branch is like plum blossom antler, the tender leaf is in a gray green color, the mature leaf is turned into a dark green color and is a main ornamental part of the platycerium, and the sporangium is scattered below the concave part of the first branch of the main lobe of the sporophyll, is green at first time and turns yellow later. The platycerium is often used as an ornamental plant hung indoors or on wall, is very popular for decoration and arrangement in places such as parks, plantations, shops, rooms, windowsills and the like in Europe and America, has the advantages of strong stress resistance, long ornamental period and the like, is a famous leaf-watching plant in the international gardening world, is a good material for indoor three-dimensional greening, is elegant and full of exotic temperament, is deeply loved by people and has wide application prospect.
The main breeding method of the platycerium is mainly based on the branch breeding and the spore breeding, but the branch breeding coefficient is small, the natural germination rate of the spores is low, and the application of the platycerium is greatly limited. The rapid propagation of the platycerium seedlings can be realized by utilizing the plant tissue culture technology, so that the platycerium seedlings are more and more emphasized by practitioners in recent years.
In 2004, Huang Jiang Ye (tassel of Huang Liang) reported that Acropodium bifidus firstly forms green spheroid (GGB) by induction with young true leaf as explant, then proliferates and differentiates bud by GGB, and finally obtains a large amount of seedlings by rooting culture, seedling hardening and transplanting. [ Huang-bang tassel ] St.bifida Botrys tissue culture [ J ]. J.Biol., 2004, 21 (5): 22-24]. In 2000, Wangzhengyi et al reported the influence of cytokinin and sucrose on the bud differentiation of Ceratopterus elaphus, and it was considered that 6-BA was suitable for the bud differentiation of adventitious buds and the amount of sugar used at 30g/L was the most preferable. Tissue culture of Wangzhenyi, Wangzying, Acropodium vulgare [ J ]. Special product of Chinese forest, 2000, 3 (54): 23-24]. In Chinese patent with application number of CN201510087218.X and named as 'a method for tissue culture and rapid propagation of platycerium' the in vitro tissue culture and rapid propagation method of platycerium is established by taking tender sporophylls of platycerium as explants and carrying out the processes of explant disinfection, GGB induction, proliferation, differentiation, rooting and the like.
Although the above documents report the tissue culture technique of platycerium, the method is limited to using young and tender true leaves as explants, and has the problems of low propagation efficiency (GGB induction rate of 50%), long seedling propagation period (more than 8 months), low rooting rate (less than 94%), and the like. The spore quantity of the platycerium is huge, the spores are attached to the leaf backs and exposed in the air, how to adopt an effective disinfection method avoids the influence on germination caused by low survival rate of the spores due to being killed by a disinfectant, how to create germination conditions to promote mass germination and enable the spores to quickly grow into seedlings, and the key for the large-scale production of the platycerium is realized. Therefore, research or optimization is carried out, so as to obtain a tissue culture rapid propagation method of the platycerium seedlings, which is relatively convenient and fast in operation process, high in germination rate, and ideal in culture efficiency and transplanting survival rate, and the method is urgently hoped by practitioners.
[ summary of the invention ]
The invention aims to solve the technical problem of providing the aseptic germination and seedling raising method for the platycerium spores, which not only simplifies the culture link, namely, the operation process is relatively convenient, but also greatly improves the germination rate, the culture efficiency and the transplanting survival rate.
The invention solves the technical problems through the following technical scheme: an aseptic germination and seedling raising method for platycerium spores comprises the following operation steps:
(1) selection and disinfection of explants: selecting a healthy stock plant as an explant material-taking object, cutting leaves with mature spores, washing the leaves clean with tap water, cutting the leaves into 5.0 multiplied by 2.0cm pieces, putting the pieces into an ultrasonic cleaner filled with sterile water, shaking and cleaning for 30-40 min, taking out the pieces, putting the pieces into a sterile container, sucking water on a superclean bench by using a sterile paper towel, and scraping the spores by using a sharp knife for later use;
(2) spore germination: inoculating the spores obtained in the step (1) into a spore germination culture medium for aseptic germination, culturing for 10-15 days, gradually turning the spores from brown to green, carrying out swelling growth to generate green spheroids (GGB) similar to orchid protocorms, culturing for 28-35 days, wherein the germination rate reaches 100%, the culture medium is not changed, the culture is continued for 30-40 days, GGB can be greatly proliferated, and the proliferation coefficient reaches 5.0-5.5;
(3) GGB differentiation culture: transferring GGB obtained in the step (2) into a GGB differentiation medium for bud differentiation culture for 50-60 days to obtain cluster bud masses with the size of 1.8-2.5 cm;
(4) rooting culture: cutting the cluster buds obtained in the step (3) into single buds, inoculating the single buds on a rooting culture medium for culturing for 70-75 days to obtain complete plants with the plant height of 5.5-6.5 cm, the root length of 2.0-3.0 cm and the number of 3-4, wherein the rooting rate is 100.0%;
(5) transplanting test-tube seedlings: before transplanting, hardening the bottle seedlings obtained in the step (4) to adapt to a cultivation environment, washing the seedlings with tap water after hardening, cleaning culture media with adhered roots, then putting the seedlings in 1.0g/L of a bactericide solution for soaking and sterilizing for 5-6 min, taking out and drying, then planting the seedlings in a plug tray filled with peat soil, and then putting the plug tray on a greenhouse shelf at the temperature of 22-28 ℃ for conventional cultivation management;
wherein, the components of the spore germination culture medium are as follows: huabao No. 1.5-2.0 g/L, KNO30.95g/L、(NH4)2SO40.85g/L、KH2PO40.25mg/L、MgSO4·7H2O 0.25g/L、CaCl2·2H2O 0.2～0.4g/L、FeSO4·7H2O27.8mg/L、Na2·EDTA 37.3mg/L、VB18.0～10.0mg/L、VB53.0～5.0mg/L、VB61.0mg/L, 2.0mg/L of glycine, 150-180 mg/L, TDZ 0.3.3-0.5 mg/L, NAA 0.1.1-0.2 mg/L of inositol, 0.5-0.8 g/L of active carbon, 30.0-35.0 g/L of white sugar and 5.6-6.0 g/L of coagulant;
GGB the components of the differentiation medium are: huabao No. 1.2-1.5 g/L, KNO30.95g/L、(NH4)2SO40.85g/L、KH2PO40.25g/L、MgSO4·7H2O 0.25g/L、CaCl2·2H2O 0.2g/L、MnSO4.H2O 16.9mg/L、ZnSO4.7H2O 8.6mg/L、H3BO36.2mg/L、KI 0.83mg/L、Na2MoO2.2H2O 0.25mg/L、CoCl2.6H2O0.025mg/L、CuSO4.5H2O 0.025mg/L、FeSO4.7H2O 27.8mg/L、Na2.EDTA 37.3mg/L、VB15.0～8.0mg/L、VB52.0～3.0mg/L、VB61.0mg/L, 2.0mg/L of glycine, 180-200 mg/L of inositol, 0.2-0.3 mg/L, KT 0.05.05-0.1 mg/L, NAA 0.2.2-0.3 mg/L, IBA 0.3-0.5 mg/L of 6-BA, 1.5-3.0 g/L of potato powder, 0.5-0.8 g/L of hydrolyzed milk protein, 30.0-35.0 g/L of white sugar and 6.0-7.2 g/L of coagulant;
the rooting medium comprises the following components: huabao No. 1.0-1.2 g/L, KNO30.65g/L、(NH4)2SO40.6g/L、KH2PO40.1g/L、MgSO4·7H2O 0.25mg/L、CaCl2·2H2O 0.2g/L、FeSO4·7H2O 27.8mg/L、Na2·EDTA 37.3mg/L、VB12.0～3.0mg/L、VB51.0～2.0mg/L、VB66.0-8.0 mg/L, 2.0-4.0 mg/L glycine, 110-130 mg/L, NAA 0.3.3-0.5 mg/L inositol, 20.0-25.0 g/L white sugar, 0.3-0.5 g/L active carbon, 3.0-4.0 g/L banana powder and 7.2-7.6 g/L coagulant.
Further, in the step (1), the ultrasonic cleaner is placed on an ultra-clean workbench before use, is sterilized by an ultraviolet lamp for 30-60 min, and then is added with sterile water. The sterilization operation of the sterile water and the sterile container is as follows: and (3) placing sterile water or a sterile container at 121 ℃ for sterilization for 30-40 min.
Further, coagulants in the germination, differentiation and rooting culture medium are all mixtures of agar powder and carrageenan, and the mass ratio of the agar powder to the carrageenan is 3: 1.
Further, the pH values of the spore germination, differentiation and rooting culture medium are 5: 8-6.0; and the culture temperature of spore germination, differentiation culture and rooting culture is (25 +/-2) DEG C, the spore germination and differentiation culture are carried out, the inoculated spore is cultured for 7-10 d in weak light, namely the natural scattered light culture in a culture room, then the spore is transferred to light culture, the light intensity is 1000-1500 lx, the illumination is 10h/d, the light intensity of rooting culture is 1800-2000 lx, and the illumination is 12 h/d.
Further, in the step (5), the hardening off is carried out in a greenhouse with a shading rate of 70-80% and a temperature of 22-28 ℃, and the closed opening is 7-10 d, the semi-open opening is 3-5 d, and the full-open opening is 2-3 d.
The invention has the beneficial effects that:
by adopting the method, only 178d to 210d is needed to obtain the platycerium seedlings; the spore germination culture medium can synchronously perform spore germination and proliferation of GGB, simplifies the culture link, reduces the culture cost, has high spore germination rate, GGB proliferation rate and short seedling forming time, namely has high propagation efficiency, and further improves the culture efficiency of the whole seedling;
in addition, the test-tube seedlings cultured by the method are strong and have developed root systems, and the environmental adaptability is strong through domestication and seedling hardening, so that the seedling transplanting survival rate is improved, and the transplanting survival rate reaches more than 98.5% in 2 months; in other words, the method overcomes the defects of relatively complicated operation process, low efficiency, poor resistance of tissue culture seedlings and low transplanting survival rate of the platycerium seedlings in the prior art.
[ detailed description ] embodiments
The invention relates to a method for aseptic germination and seedling raising of platycerium spores, which comprises the following specific operation steps:
(1) selection and disinfection of explants: selecting a healthy stock plant as an explant material-taking object, cutting leaves with mature spores, washing the leaves clean with tap water, cutting the leaves into 5.0 multiplied by 2.0cm pieces, putting the pieces into an ultrasonic cleaner filled with sterile water, shaking and cleaning for 30-40 min, taking out the pieces, putting the pieces into a sterile container, sucking water on a superclean bench by using a sterile paper towel, and scraping the spores by using a sharp knife for later use.
(2) Spore germination: and (2) inoculating the spores obtained in the step (1) into a spore germination culture medium for aseptic germination, culturing for 10-15 days, gradually turning the spores from brown to green, performing swelling growth to generate green spheroids (GGB) similar to orchid protocorms, culturing for 28-35 days, wherein the germination rate reaches 100%, and continuously culturing for 30-40 days without replacing the culture medium, wherein GGB can be greatly proliferated, and the proliferation coefficient reaches 5.0-5.5.
(3) GGB differentiation culture: and (3) transferring GGB obtained in the step (2) into a GGB differentiation medium for bud differentiation culture, and culturing for 50-60 days to obtain clump buds with the size of 1.8-2.5 cm.
(4) Rooting culture: cutting the cluster buds obtained in the step (3) into single buds, inoculating the single buds on a rooting culture medium for culturing for 70-75 days to obtain complete plants with the plant height of 5.5-6.5 cm, the root length of 2.0-3.0 cm and the number of 3-4, wherein the rooting rate is 100.0%;
(5) transplanting test-tube seedlings: before transplanting, hardening the bottle seedlings obtained in the step (4) to be adaptive to a cultivation environment, washing the seedlings with tap water after hardening, cleaning culture media with adhered roots, then putting the seedlings in 1.0g/L of a bactericide solution for soaking and sterilizing for 5-6 min, taking out and drying, then planting the seedlings in a plug tray filled with peat soil, and then putting the plug tray on a greenhouse shelf at the temperature of 22-28 ℃ for conventional cultivation management.
Wherein, the components of the spore germination culture medium are as follows: huabao No. 1.5-2.0 g/L, KNO30.95g/L、(NH4)2SO40.85g/L、KH2PO40.25mg/L、MgSO4·7H2O 0.25g/L、CaCl2·2H2O 0.2～0.4g/L、FeSO4·7H2O27.8mg/L、Na2·EDTA 37.3mg/L、VB18.0～10.0mg/L、VB53.0～5.0mg/L、VB61.0mg/L, 2.0mg/L of glycine, 150-180 mg/L, TDZ 0.3.3-0.5 mg/L, NAA 0.1.1-0.2 mg/L of inositol, 0.5-0.8 g/L of active carbon, 30-35 g/L of white sugar and 5.6-6.0 g/L of coagulant;
GGB the components of the differentiation medium are: huabao No. 1.2-1.5 g/L, KNO30.95g/L、(NH4)2SO40.85g/L、KH2PO40.25g/L、MgSO4·7H2O 0.25g/L、CaCl2·2H2O 0.2g/L、MnSO4.H2O 16.9mg/L、ZnSO4.7H2O 8.6mg/L、H3BO36.2mg/L、KI 0.83mg/L、Na2MoO2.2H2O 0.25mg/L、CoCl2.6H2O0.025mg/L、CuSO4.5H2O 0.025mg/L、FeSO4.7H2O 27.8mg/L、Na2.EDTA 37.3mg/L、VB15.0～8.0mg/L、VB52.0～3.0mg/L、VB61.0mg/L, 2.0mg/L of glycine, 180-200 mg/L of inositol, 0.2-0.3 mg/L, KT 0.05.05-0.1 mg/L, NAA 0.2.2-0.3 mg/L, IBA 0.3-0.5 mg/L of 6-BA, 1.5-3.0 g/L of potato powder, 0.5-0.8 g/L of hydrolyzed milk protein, 30.0-35.0 g/L of white sugar and 6.0-7.2 g/L of coagulant;
the rooting medium comprises the following components: huabao No. 1.0-1.2 g/L, KNO30.65g/L、(NH4)2SO40.6g/L、KH2PO40.1g/L、MgSO4·7H2O 0.25mg/L、CaCl2·2H2O 0.2g/L、FeSO4·7H2O 27.8mg/L、Na2·EDTA 37.3mg/L、VB12.0～3.0mg/L、VB51.0～2.0mg/L、VB66.0-8.0 mg/L, 2.0-4.0 mg/L glycine, 110-130 mg/L, NAA 0.3.3-0.5 mg/L inositol, 20.0-25.0 g/L white sugar, 0.3-0.5 g/L active carbon, 3.0-4.0 g/L banana powder and 7.2-7.6 g/L coagulant.
In the specific embodiment of the invention, coagulants of the spore germination, differentiation and rooting culture medium can adopt a mixture of agar powder and carrageenan, and the mass ratio of the agar powder to the carrageenan is 3:1, the cost is lower. The pH values of spore germination, differentiation and rooting culture media are 5.8-6.0; and the culture temperature of spore germination, differentiation culture and rooting culture is (25 +/-2) DEG C, the spore germination and differentiation culture are carried out, the inoculated spore is cultured for 7-10 d in weak light, namely the natural scattered light culture in a culture room, then the spore is transferred to light culture, the light intensity is 1000-1500 lx, the illumination is 10h/d, the light intensity of rooting culture is 1800-2000 lx, and the illumination is 12 h/d.
In addition, unless otherwise specified, the percentages in the present invention are all mass percentages. In the invention, the mass ratio of N, P, K in the American producing area of Huabao 1 is 7: 6: 19; agar powder and carrageenan in Japan, the strength is 1400g/cm2、1500g/cm2(ii) a The white sugar is bagged white sugar with the first-grade quality on the market.
For further illustration of the method of the present invention, the following examples are given by the applicant and are only illustrative and not intended to limit the scope of the present invention.
Example one
Selection and disinfection of explants: selecting healthy mother plants of Ceratopteris dichotoma as explant material-taking objects, cutting leaves with mature spores, washing with tap water, cutting the leaves into pieces with size of 5.0 × 2.0cm, washing in an ultrasonic cleaner filled with sterile water for 30min, taking out, placing in a sterile container, sucking water on a superclean bench with sterile paper towel, and scraping off the spores with a sharp knife for later use.
Spore germination: clamping spores by using tweezers and uniformly scattering the spores on the surface of a spore germination culture medium for culturing; the spore is sowed and cultured for 10 days, the color of the spore is gradually changed to green, the spore grows in an expanding way, the seed is inoculated for 28 days and germinates into green spheroids (GGB) (the germination rate is up to 100.0 percent), then GGB is proliferated in a large quantity, the seed is inoculated for 30 days, the bottom of the bottle is full of GGB lumps of dense ramie, and the proliferation coefficient is up to 5.0; the components of the spore germination culture medium are as follows: huabao No. 1.5g/L, KNO30.95g/L、(NH4)2SO40.85g/L、KH2PO40.25mg/L、MgSO4·7H2O 0.25g/L、CaCl2·2H2O 0.2g/L、FeSO4·7H2O 27.8mg/L、Na2·EDTA37.3mg/L、VB18.0mg/L、VB53.0mg/L、VB61.0mg/L, 2.0mg/L glycine, 150mg/L inositol, 0.1mg/L TDZ0.3mg/L, NAA 0.1, 0.5g/L active carbon, 30.0g/L white sugar and 5.6g/L coagulant.
GGB differentiation culture: GGB is transferred into a differentiation medium for bud differentiation culture, and a cluster bud mass with the size of 1.8-2.0 cm can be obtained after the culture period of 50 d; the components of the differentiation medium are: huabao No. 1.2g/L, KNO30.95g/L、(NH4)2SO40.85g/L、KH2PO40.25g/L、MgSO4·7H2O 0.25g/L、CaCl2·2H2O 0.2g/L、MnSO4.H2O 16.9mg/L、ZnSO4.7H2O 8.6mg/L、H3BO36.2mg/L、KI 0.83mg/L、Na2MoO2.2H2O 0.25mg/L、CoCl2.6H2O0.025mg/L、CuSO4.5H2O 0.025mg/L、FeSO4.7H2O 27.8mg/L、Na2.EDTA 37.3mg/L、VB15.0mg/L、VB52.0mg/L、VB61.0mg/L, 2.0mg/L glycine, 180mg/L inositol, 0.2mg/L, KT 0.05.05 mg/L, NAA 0.2.2 mg/L, IBA 0.3.3 mg/L6-BA, 1.5g/L potato powder, 0.5g/L hydrolyzed milk protein, 30.0g/L white sugar and 6.0g/L coagulant.
Rooting culture: inoculating the obtained robust plantlets into a rooting culture medium for culturing, and culturing for 70 days to obtain complete plants, namely seedlings (the rooting rate reaches 100.0%, and the seedlings are robust), wherein the plant height is 5.5-6.0 cm, the root length is 2.0-2.5 cm, and the number of the roots is 3-4; the rooting medium comprises the following components: huabao No. 1.0g/L, KNO30.65g/L、(NH4)2SO40.6g/L、KH2PO40.1g/L、MgSO4·7H2O 0.25mg/L、CaCl2·2H2O 0.2g/L、FeSO4·7H2O 27.8mg/L、Na2·EDTA37.3mg/L、VB12.0mg/L、VB51.0mg/L、VB66.0mg/L, 2.0mg/L glycine, 110mg/L, NAA 0.3.3 mg/L inositol, 20.0g/L white sugar, 0.3g/L active carbon, 3.0g/L banana powder and 7.2g/L coagulant.
Transplanting test-tube seedlings: before transplanting, hardening seedlings obtained after rooting culture to be adaptive to a culture environment, washing the seedlings by using tap water after hardening, cleaning a culture medium adhered to the roots of the seedlings, then putting the seedlings into a carbendazim solution with the concentration of 1.0g/L for soaking and sterilizing for 5min, taking out the seedlings, airing the seedlings, then planting the seedlings into a plug tray filled with peat soil, then putting the plugs on a greenhouse shelf for conventional culture management, and transplanting the seedlings to reach 98.0% in 2 months.
Example two
Selection and disinfection of explants: selecting healthy mother plants of platycerium wallichii as explant material-taking objects, cutting leaves with mature spores, washing the leaves with tap water, cutting the leaves into 5.0 multiplied by 2.0cm pieces, placing the pieces into an ultrasonic cleaner filled with sterile water, shaking and cleaning the pieces for 35min, taking the pieces out, placing the pieces into a sterile container, sucking water on a superclean workbench by using a sterile paper towel, and scraping the spores by using a sharp knife for later use.
Spore germination: clamping spores by using tweezers and uniformly scattering the spores on the surface of a spore germination culture medium for culturing; the spores are sown and cultured for 12d, the color turns green gradually, and the spores grow in an expanding way, and are inoculated for 30d to germinate into green spheroids (GGB) (the germination rate is as high as 100.0 percent), then GGB is proliferated in a large quantity, and 35d is inoculated, the bottle bottom is full of GGB lumps of dense ramie, and the proliferation coefficient is 5.2; the components of the spore germination culture medium are as follows: huabao No. 1.8g/L, KNO30.95g/L、(NH4)2SO40.85g/L、KH2PO40.25mg/L、MgSO4·7H2O 0.25g/L、CaCl2·2H2O 0.3g/L、FeSO4·7H2O 27.8mg/L、Na2·EDTA37.3mg/L、VB19.0mg/L、VB54.0mg/L、VB61.0mg/L, 2.0mg/L glycine, 160mg/L inositol, 0.4mg/L, NAA 0.2mg/L TDZ0.2 mg/L active carbon 0.6g/L white sugar 33.0g/L coagulant 5.8 g/L.
GGB differentiation culture: GGB is transferred into a differentiation medium for bud differentiation culture, and a cluster bud mass with the size of 1.8-2.2 cm can be obtained in a culture period of 55 d; the components of the differentiation medium are: huabao No. 1.3g/L, KNO30.95g/L、(NH4)2SO40.85g/L、KH2PO40.25g/L、MgSO4·7H2O 0.25g/L、CaCl2·2H2O 0.2g/L、MnSO4.H2O 16.9mg/L、ZnSO4.7H2O 8.6mg/L、H3BO36.2mg/L、KI 0.83mg/L、Na2MoO2.2H2O 0.25mg/L、CoCl2.6H2O0.025mg/L、CuSO4.5H2O 0.025mg/L、FeSO4.7H2O 27.8mg/L、Na2.EDTA 37.3mg/L、VB16.0mg/L、VB52.5mg/L、VB61.0mg/L, 2.0mg/L glycine, 190mg/L inositol, 0.3mg/L, KT 0.1.1 mg/L, NAA 0.3.3 mg/L, IBA 0.4.4 mg/L6-BA, 2.0g/L potato powder, 0.6g/L hydrolyzed milk protein, 33.0g/L white sugar and 6.6g/L coagulant.
Rooting cultureCulturing: inoculating the obtained robust plantlets into a rooting culture medium for culturing, and culturing for 72 days to obtain complete plants, namely seedlings (the rooting rate reaches 100.0%, and the seedlings are robust), wherein the plant height is 5.8-6.3 cm, the root length is 2.5-3.0 cm, and the number of the roots is 3-4; the rooting medium comprises the following components: huabao No. 1.1g/L, KNO30.65g/L、(NH4)2SO40.6g/L、KH2PO40.1g/L、MgSO4·7H2O 0.25mg/L、CaCl2·2H2O 0.2g/L、FeSO4·7H2O 27.8mg/L、Na2·EDTA37.3mg/L、VB12.5mg/L、VB51.5mg/L、VB67.0mg/L, 3.0mg/L glycine, 120mg/L, NAA 0.4.4 mg/L inositol, 23.0g/L white sugar, 0.4g/L active carbon, 3.5g/L banana powder and 7.4g/L coagulant.
Transplanting test-tube seedlings: before transplanting, hardening seedlings obtained after rooting culture to be adaptive to a cultivation environment, washing the seedlings by using tap water after hardening, cleaning a culture medium adhered to the roots of the seedlings, then putting the seedlings into a chlorothalonil solution with the concentration of 1.0g/L for soaking and sterilizing for 6min, taking out the seedlings, airing the seedlings, then planting the seedlings into a plug tray filled with peat soil, then putting the plugs on a greenhouse shelf for conventional cultivation management, and transplanting the seedlings to reach 98.5% in 2 months.
EXAMPLE III
Selection and disinfection of explants: selecting healthy mother plants of Ceratopteris dichotoma as explant material-taking objects, cutting leaves with mature spores, washing with tap water, cutting the leaves into pieces with size of 5.0 × 2.0cm, placing into an ultrasonic cleaner filled with sterile water, shaking and cleaning for 40min, taking out, placing into a sterile container, sucking water on a superclean bench with sterile paper towel, and scraping off the spores with a sharp knife for later use.
Spore germination: clamping spores by using tweezers and uniformly scattering the spores on the surface of a spore germination culture medium for culturing; the spores are sown and cultured for 15 days, the color turns green gradually from brown, and the spores grow in an expanding way, 35 days of inoculation germinate into green spheroids (GGB) (the germination rate is up to 100.0 percent), then GGB proliferates in a large quantity, 40 days of inoculation are carried out, GGB lumps of dense ramie grow on the bottoms of bottles, and the proliferation coefficient reaches 5.5; the components of the spore germination culture medium are as follows: huabao No. 1 No. 2.0g/L, KNO30.95g/L、(NH4)2SO40.85g/L、KH2PO40.25mg/L、MgSO4·7H2O 0.25g/L、CaCl2·2H2O 0.4g/L、FeSO4·7H2O 27.8mg/L、Na2·EDTA37.3mg/L、VB110.0mg/L、VB55.0mg/L、VB61.0mg/L, 2.0mg/L glycine, 180mg/L inositol, TDZ0.5mg/L, NAA 0.2.2 mg/L, 0.8g/L active carbon, 35.0g/L white sugar and 6.0g/L coagulant.
GGB differentiation culture: transferring GGB into a differentiation culture medium for bud differentiation culture, and culturing for 60 days to obtain cluster bud masses with the size of 2.2-2.5 cm; the components of the differentiation medium are: huabao No. 1.5g/L, KNO30.95g/L、(NH4)2SO40.85g/L、KH2PO40.25g/L、MgSO4·7H2O 0.25g/L、CaCl2·2H2O 0.2g/L、MnSO4.H2O 16.9mg/L、ZnSO4.7H2O 8.6mg/L、H3BO36.2mg/L、KI 0.83mg/L、Na2MoO2.2H2O 0.25mg/L、CoCl2.6H2O0.025mg/L、CuSO4.5H2O 0.025mg/L、FeSO4.7H2O 27.8mg/L、Na2.EDTA 37.3mg/L、VB15.0～8.0mg/L、VB53.0mg/L、VB61.0mg/L, 2.0mg/L of glycine, 180-200 mg/L of inositol, 0.3mg/L of 6-BA, 0.1mg/L, NAA 0.3.3 mg/L, IBA 0.5.5 mg/L of KT, 3.0g/L of potato powder, 0.8g/L of hydrolyzed milk protein, 35.0g/L of white sugar and 7.2g/L of coagulant.
Rooting culture: inoculating the obtained robust plantlets into a rooting culture medium for culturing, and culturing for 75 days to obtain complete plants, namely seedlings (the rooting rate reaches 100.0%, and the seedlings are robust), wherein the plant height is 6.0-6.5 cm, the root length is 2.0-3.0 cm, and the number of the roots is 3-4; the rooting medium comprises the following components: huabao No. 1.2g/L, KNO30.65g/L、(NH4)2SO40.6g/L、KH2PO40.1g/L、MgSO4·7H2O 0.25mg/L、CaCl2·2H2O 0.2g/L、FeSO4·7H2O 27.8mg/L、Na2·EDTA37.3mg/L、VB13.0mg/L、VB52.0mg/L、VB68.0mg/L, 4.0mg/L glycine, 130mg/L inositol, 0.5mg/L NAA0, 25.0g/L white sugar, 0.5g/L active carbon, 4.0g/L banana powder and 7.6g/L coagulant.
Transplanting test-tube seedlings: before transplanting, hardening seedlings obtained after rooting culture to be adaptive to a culture environment, washing the seedlings by using tap water after hardening, cleaning a culture medium adhered to the roots of the seedlings, then putting the seedlings into a carbendazim solution with the concentration of 1.0g/L for soaking and sterilizing for 6min, taking out the seedlings, airing the seedlings, then planting the seedlings into a plug tray filled with peat soil, then putting the plugs on a greenhouse shelf for conventional culture management, and transplanting the seedlings with the survival rate of 99.0 percent in 2 months.
In conclusion, by adopting the method, only 178d to 210d is needed to obtain the platycerium seedlings; the spore germination culture medium can synchronously perform spore germination and proliferation of GGB, simplifies the culture link, reduces the culture cost, has high spore germination rate, GGB proliferation rate and short seedling forming time, namely has high propagation efficiency, and further improves the culture efficiency of the whole seedling;
in addition, the test-tube seedlings cultured by the method are strong and have developed root systems, and the environmental adaptability is strong through domestication and seedling hardening, so that the seedling transplanting survival rate is improved, and the transplanting survival rate reaches more than 98.5% in 2 months; in other words, the method overcomes the defects of relatively complicated operation process, low efficiency, poor resistance of tissue culture seedlings and low transplanting survival rate of the platycerium seedlings in the prior art.
Although specific embodiments of the invention have been described above, it will be understood by those skilled in the art that the specific embodiments described are illustrative only and are not limiting upon the scope of the invention, and that equivalent modifications and variations can be made by those skilled in the art without departing from the spirit of the invention, which is to be limited only by the appended claims.
Claims (5)
1. An aseptic germination seedling raising method for platycerium spores is characterized by comprising the following steps: the method comprises the following operation steps:
(1) selection and disinfection of explants: selecting a healthy stock plant as an explant material taking object, cutting leaves with mature spores, washing, cutting the leaves into pieces, putting the pieces into an ultrasonic cleaner filled with sterile water, shaking and cleaning for 30-40 min, taking out the pieces, putting the pieces into a sterile container, sucking water on an ultra-clean workbench by using a sterile paper towel, and scraping the spores by using a sharp knife for later use;
(2) spore germination: inoculating the spores obtained in the step (1) into a spore germination culture medium for aseptic germination, culturing for 10-15 days, gradually turning the spores from brown to green, carrying out swelling growth to generate green spheroids GGB similar to orchid protocorms, culturing for 28-35 days, carrying out spore germination, continuously culturing for 30-40 days without changing the culture medium, carrying out mass proliferation on GGB, and enabling the proliferation coefficient to reach 5.0-5.5;
(3) GGB differentiation culture: GGB obtained in the step (2) is transferred into a GGB differentiation medium for bud differentiation culture, and the bud differentiation culture is carried out for 50-60 days to obtain a cluster bud mass;
(4) rooting culture: cutting the cluster buds obtained in the step (3) into single buds, inoculating the single buds on a rooting culture medium for culturing for 70-75 days to obtain a complete plant;
(5) transplanting test-tube seedlings: before transplanting, hardening the bottle seedlings obtained in the step (4) to adapt to a cultivation environment, washing the seedlings after hardening, cleaning culture media with adhered roots, then putting the seedlings in a bactericide solution for soaking and sterilizing for 5-6 min, taking out and drying in the air, then planting the seedlings in a plug tray filled with peat soil, and then putting the plug tray on a greenhouse shelf at 22-28 ℃ for conventional cultivation management;
wherein, the components of the spore germination culture medium are as follows: huabao No. 1.5-2.0 g/L, KNO30.95g/L、(NH4)2SO40.85g/L、KH2PO40.25mg/L、MgSO4·7H2O 0.25g/L、CaCl2·2H2O 0.2～0.4g/L、FeSO4·7H2O27.8mg/L、Na2·EDTA 37.3mg/L、VB18.0～10.0mg/L、VB53.0～5.0mg/L、VB61.0mg/L, 2.0mg/L of glycine, 150-180 mg/L, TDZ 0.3.3-0.5 mg/L, NAA 0.1.1-0.2 mg/L of inositol, 0.5-0.8 g/L of active carbon, 30.0-35.0 g/L of white sugar and 5.6-6.0 g/L of coagulant;
GGB the components of the differentiation medium are: huabao No. 1.2-1.5 g/L, KNO30.95g/L、(NH4)2SO40.85g/L、KH2PO40.25g/L、MgSO4·7H2O 0.25g/L、CaCl2·2H2O 0.2g/L、MnSO4.H2O 16.9mg/L、ZnSO4.7H2O 8.6mg/L、H3BO36.2mg/L、KI 0.83mg/L、Na2MoO2.2H2O 0.25mg/L、CoCl2.6H2O0.025mg/L、CuSO4.5H2O 0.025mg/L、FeSO4.7H2O 27.8mg/L、Na2.EDTA 37.3mg/L、VB15.0～8.0mg/L、VB52.0～3.0mg/L、VB61.0mg/L, 2.0mg/L of glycine, 180-200 mg/L of inositol, 0.2-0.3 mg/L, KT 0.05.05-0.1 mg/L, NAA 0.2.2-0.3 mg/L, IBA 0.3-0.5 mg/L of 6-BA, 1.5-3.0 g/L of potato powder, 0.5-0.8 g/L of hydrolyzed milk protein, 30.0-35.0 g/L of white sugar and 6.0-7.2 g/L of coagulant;
the rooting medium comprises the following components: huabao No. 1.0-1.2 g/L, KNO30.65g/L、(NH4)2SO40.6g/L、KH2PO40.1g/L、MgSO4·7H2O 0.25mg/L、CaCl2·2H2O 0.2g/L、FeSO4·7H2O 27.8mg/L、Na2·EDTA37.3mg/L、VB12.0～3.0mg/L、VB51.0～2.0mg/L、VB66.0-8.0 mg/L, 2.0-4.0 mg/L glycine, 110-130 mg/L, NAA 0.3.3-0.5 mg/L inositol, 20.0-25.0 g/L white sugar, 0.3-0.5 g/L active carbon, 3.0-4.0 g/L banana powder and 7.2-7.6 g/L coagulant.
2. The aseptic germination seedling raising method of platycerium wallichii spores according to claim 1, which is characterized in that: in the step (1), the ultrasonic cleaner is placed on an ultra-clean workbench before use, is sterilized by an ultraviolet lamp for 30-60 min, and then is added with sterile water; the sterilization operation of the sterile water and the sterile container is as follows: and (3) placing sterile water or a sterile container at 121 ℃ for sterilization for 30-40 min.
3. The aseptic germination seedling raising method of platycerium wallichii spores according to claim 1, which is characterized in that: the coagulants in the spore germination, differentiation and rooting culture medium are mixtures of agar powder and carrageenan, and the mass ratio of the agar powder to the carrageenan is 3: 1.
4. The aseptic germination seedling raising method of platycerium wallichii spores according to claim 1, which is characterized in that: the pH value of the germination, differentiation and rooting culture medium is 5: 8-6.0;
and the culture temperature of spore germination, differentiation culture and rooting culture is 25 +/-2 ℃, the spore germination and differentiation culture is carried out, the inoculated spore is cultured for 7-10 d in weak light, namely the natural scattering light culture in a culture room, then the spore is transferred to light culture, the light intensity is 1000-1500 lx, the illumination is 10h/d, the light intensity of rooting culture is 1800-2000 lx, and the illumination is 12 h/d.
5. The aseptic germination seedling raising method of platycerium wallichii spores according to claim 1, which is characterized in that: in the step (5), the hardening off is carried out in a greenhouse with a shading rate of 70-80% and a temperature of 22-28 ℃, and the closed opening is 7-10 d, the semi-open opening is 3-5 d, and the full-open opening is 2-3 d.
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