CN113475189A - Orchid plant seed-fungus symbiotic germination complex and preparation method and application thereof - Google Patents
Orchid plant seed-fungus symbiotic germination complex and preparation method and application thereof Download PDFInfo
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Abstract
The invention provides an orchid seed-fungus symbiotic germination complex as well as a preparation method and application thereof, and belongs to the technical field of biology. The preparation method of the orchid seed-fungus symbiotic germination complex comprises the steps of preparing seed-fungus mixed suspension from seeds and fungus mycelia, dripping the suspension into sodium alginate aqueous solution to obtain an embedding body, and hardening the embedding body to obtain the seed-fungus symbiotic germination complex. The complex is spherical, and each complex contains 5-10 seeds. The seeds germinate and form seedlings about 30 days after the complex is sowed, the germination rate and the seedling survival rate are high, the complex can be directly applied to sowing and large-scale centralized seedling raising under natural conditions, and the complex has the advantages of simple manufacturing process, low cost, commercial production, convenient storage, transportation and use, high seed germination rate, quick seedling formation and the like.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to an orchid seed-fungus symbiotic germination complex as well as a preparation method and application thereof.
Background
The orchid family (Orchidaceae) is one of the largest families of angiosperms, and is about 800 genus 28000 species, but is also the most threatened plant group in the whole sphere. China is one of the most abundant countries of the orchid family, and is 1700, but the species close to 1/3 are in danger because of high ornamental or medicinal value and are excessively collected for a long time. The dendrobe (Dendrobium) is the second largest genus of Orchidaceae, about 1500-1600 species are epiphytes throughout the world, and are mainly distributed in southeast Asia of tropical zone and oceania region. More than 80 kinds of dendrobium plants exist in China, nearly half of the kinds of dendrobium plants are used for traditional Chinese medicinal materials and health care products, and the dendrobium plants have a long utilization history. Due to the long-term excessive collection of wild dendrobium resources, many dendrobium plants are seriously threatened, and the protection and sustainable utilization of the dendrobium plants are urgently needed to be developed.
Orchidaceae plants produce fine and large quantities of "dust" seeds, which lack endosperm, and naturally, the seeds need to rely on specific mycorrhizal fungi to provide nutrition to promote germination and seedling formation, and often need to rely on symbiotic fungi during growth and development after germination, and only after the fungi and the roots of seedlings form symbiotic mycorrhiza, the absorption and utilization of water and mineral nutrition are improved, so that the plants can normally grow and develop (Arditti et al, 2000; Smith et al, 2008; Martin, 2016). The mycorrhizal fungi is utilized to promote the seed germination and obtain seedlings, the survival rate and the growth speed of the seedlings in the natural environment can be obviously improved, and the mycorrhizal fungi is considered as an important way for protecting endangered orchids (Stewart et al, 2003; Batty et al, 2006; Otero et al, 2013).
In the past 30 years, due to the huge market demand on medicinal dendrobium, and the maturity and application of the non-symbiotic germination (aseptic germination) technology of the seeds of the orchidaceae plants are carried out by utilizing the artificial culture medium, so that the large-scale propagation of seedlings by utilizing the seeds is realized, the rapid development of the medicinal dendrobium industry which takes the orchidaceae plants (D.officinale) as main cultivation varieties, produces the seedlings by aseptic germination of the seeds and takes greenhouse intensive cultivation as a main mode is promoted. Because the product quality (drug effect) of the medicinal dendrobium lacks of a unified standard, consumers have concerns about the drug effect and safety of dendrobium products in an intensive cultivation mode, and common consumers generally consider that the wild drug effect is better and safer in the consumption psychology of Chinese medicinal materials and health care products. The development of ecological cultivation of medicinal dendrobium under natural conditions has also become an important direction for the consensus and industrial sustainability and healthy development in the dendrobium industry (Cheng et al, 2019).
The change of the cultivation mode from greenhouse intensive cultivation to ecological cultivation under natural conditions of the medicinal dendrobium inevitably leads to the change of the aspects of production and supply of seedlings, cultivation management technology and the like. On the premise of obtaining effective mycorrhizal fungi for germination of specific medicinal dendrobium seeds, the mycorrhizal fungi are utilized to carry out symbiotic germination of seeds, the production cost of seedlings can be greatly reduced, links such as seedling exercising and transplanting are not needed, the seeds germinate, the seedlings are formed, the seedlings grow to adult plants under natural conditions, the product can completely meet the requirement on the wild characteristic of the medicinal dendrobium, and the method is very suitable for ecological cultivation of the medicinal dendrobium.
However, the orchid seed has a certain selectivity for germinating fungi and has specific requirements for germination conditions, so that there are many problems in natural symbiosis of the orchid seed and the fungi in nature, such as high uncertainty in distribution and existence of specific fungi under natural conditions, incapability of maintaining stable symbiotic conditions in nature, and the like, which all result in very low germination rate and seedling rate of the orchid seed.
In order to overcome the problem of natural symbiosis, the prior art prepares an artificial seed of an orchid plant, and protocorm formed by aseptic germination is mixed with artificial culture medium components (a plurality of inorganic salt major elements, trace elements, organic matters, phytohormones and the like) to be embedded to prepare the artificial seed (Vannoff 2014; Wuwenjie 2006). Such artificial seeds are equivalent to a micro-scale sterile in vitro culture system, and protocorms are continuously differentiated into seedlings in a culture medium, but the manufacturing operation process is easy to pollute and cause the death of the protocorms. In aseptic germination system, rely on the culture medium to provide nutrition in the orchid seed blake bottle and germinate, form protocorm, the differentiation becomes the seedling, needs longer cycle, still needs the switching after the seedling grows to the certain degree, and the seedling goes out the bottle at last, washs and practises the seedling, just can transplant, and in small artifical seed, whether this process can accomplish smoothly, forms healthy seedling degree of difficulty very big.
Reference documents:
Arditti J,GhaniAKA.Numerical andphysical properties oforchid seeds andtheirbiological implications[J].New Phytol,2000,145:367-421.
Batty A L,Brundrett M C,Dixon K W,et al.New methods to improve symbiotic propagation of temperate terrestrial orchid seedlings from axenic culture to soil[J].Aust J Bot,2006,54(4):367-374.
Cheng J,Dang P P,Zhao Z,et al.An assessment of the Chinese medicinal Dendrobium industry:Supply,demand and sustainability[J].J Ethnopharmacol,2019,229:81-88.
Martin F.Molecular Mycorrhizal Symbiosis[M].New Jersey:Wiley-Blackwell,2016:63-86.
Otero J T,Mosquera A T,Flanagan N S.Tropical orchid mycorrhizae:potential applications in orchid conservation,commercialization,and beyond[J].Lankesteriana 2013,13(1-2):57-63.
Smith S E,Read D J.Mycorrhizal Symbiosis.3rd[M].San Diego:Academic Press,2008.
Stewart S L,Zettler L W,Minso J,et al.Symbiotic germination and reintroduction of Spiranthes brevilabris Lindley,an endangered orchid native to Florida[J].Selbyana,2003:64-70.
van Tengfei, an orchid artificial seed industrialized production key technology [ D ]. university of Dalian Industrial, 2014.
Wuvingjie, Oncidium (oncodium) protocorm liquid proliferation and artificial seed production research [ D ]. Nanjing agriculture university, 2006.
Disclosure of Invention
In view of the above, the present invention provides an orchid seed-fungus symbiotic germination complex, and a preparation method and an application thereof, wherein the orchid seed-fungus symbiotic germination complex can simulate a germination process of seeds under natural conditions, and simultaneously ensure that the seeds obtain high germination rate and seedling rate.
The invention provides a preparation method of an orchid seed-fungus symbiotic germination complex, which is characterized by comprising the following steps:
1) preparing hypha of symbiotic fungi into a fungus hypha suspension;
2) mixing the fungal hypha suspension obtained in the step 1) with the orchid seeds to obtain an orchid seed-fungal mixed suspension;
3) mixing the mixed suspension of the seeds of the orchidaceae and the fungi in the step 2) with an aqueous solution of sodium alginate to obtain an orchidaceae seed-fungi embedded body;
4) dripping the orchid seed-fungus embedding body in the step 3) into a calcium chloride aqueous solution for ion exchange reaction to obtain an orchid seed-fungus symbiotic germination complex.
Preferably, the symbiotic fungi include the order Ceratophylla (Sebacinales), Mycolepsylliaceae (Tulasnellaceae) or Ceratobasidaceae (Ceratobasidioceae);
the Orchidaceae plant includes Dendrobium (Dendrobium), Bletilla (Bletilla), Paphiopedilum (Paphiopedilum) and/or Cymbidium (Cymbium).
Preferably, the fungus of the order ceriferales comprises Sebacinales LQ or Pilus indica DSM 11827;
the plant of the genus Dendrobium includes Dendrobium officinale (Dendrobium officinale Kimura et Migo).
Preferably, the mass ratio of the fungal hyphae to the orchid seeds in the fungal hypha suspension in the step 2) is 20 (0.13-0.26);
the density of the orchid seeds in the orchid seed-fungus mixed suspension in the step 2) is 25-50 grains/ml.
Preferably, the volume ratio of the mixed suspension of the seed-fungus of the orchid family plant in the step 3) to the sodium alginate aqueous solution is (1.8-2.2): 1;
the mass percentage of the sodium alginate aqueous solution is 2.5-3.5%.
Preferably, the dosage of the orchid seed-fungus embedding body in the step 4) is 9-12 ml per time.
The invention provides an orchid seed-fungus symbiotic germination complex prepared by the preparation method, which comprises calcium alginate hydrogel and a mixture of orchid seed and mycelial of symbiotic fungus embedded in the calcium alginate hydrogel.
Preferably, each orchid seed-fungus symbiotic germination complex comprises 5-10 orchid seeds;
the mass ratio of the hypha of the symbiotic fungi to the seeds of the orchids is 20 (0.13-0.26).
The invention provides an application of the orchid seed-fungus symbiotic germination complex in orchid cultivation.
The orchid seed-fungus symbiotic germination complex provided by the invention comprises calcium alginate hydrogel and a mixture of orchid seed and mycelial of symbiotic fungus embedded in the calcium alginate hydrogel. The invention creatively mixes and embeds the seeds of the orchids and the symbiotic fungi to prepare the orchids seed-fungi symbiotic germination complex, and completely follows the principle that the seeds of the orchids germinate under natural conditions. Since the orchid plants produce a large amount of seeds to search specific germination fungi and suitable conditions, the distribution and existence of the specific fungi under natural conditions have high uncertainty, the seed germination rate and seedling rate are very low, and the seed-fungus symbiotic germination complex is equivalent to 'helping' seeds find matched specific fungi, so that the extremely high seed germination rate and seedling formation rate can be obtained. In actual use, the seed-fungus symbiotic germination composite can be directly sown under natural conditions, the seeds can quickly germinate to form seedlings after sowing, and the seedlings can well adapt to the natural environment;
the invention also provides a preparation method of the orchid seed-fungus symbiotic germination complex, the orchid seed-fungus symbiotic germination complex is directly used as a propagule for embedding, compared with the prior artificial seed technology in which the original bulbodium obtained by induction is used as an embedding medium, the method has the advantages that the operation is simple, the complicated process of inducing the original bulbum is simplified, and the preparation time is greatly shortened; the method of embedding protocorms is difficult to store because protocorms are easily contaminated, grow rapidly at normal temperature or freeze easily at low temperature. The invention directly embeds the seeds and the symbiotic fungi, thereby being convenient for storage and transportation; meanwhile, the symbiotic relationship between orchid and fungi is preliminarily established, the germination rate and the seedling rate of seeds are improved, the seeds are easy to germinate when being sowed in a natural environment, the environment adaptability is good, and a new path is opened for carrying out symbiotic germination and seedling culture of seeds by utilizing the fungi. Compared with the tissue culture technology, the method has the advantages of low cost, low requirements on technology and facilities, easy operation, small volume and quick germination and seedling of seeds; can efficiently and quickly obtain a large amount of dendrobium officinale seed-fungus symbiotic germination complexes in a short time, is convenient to store and transport, can be directly sowed under natural conditions, and the like, and is favorable for realizing large-scale seedling production and regression protection of orchid plant resources. The method has important theoretical significance and practical application value for the artificial cultivation and field regression of rare and endangered orchids which are difficult to propagate, or the ecological cultivation and concentrated seedling raising of medicinal orchids and the like.
Drawings
FIG. 1 is a morphological diagram of a ferric sheet seed-fungus symbiotic germination complex prepared by the invention;
FIG. 2 shows the germination of seeds and the growth of seedlings of the symbiotic germination complex of Dendrobium officinale seeds and fungi of the present invention after seeding for 30 days and 60 days, wherein FIG. 2A shows the germination of the symbiotic germination complex of Dendrobium officinale seeds and fungi on oat medium and mixed medium for 30 days; FIG. 2B shows the germination of the Dendrobium officinale seed-fungus symbiotic germination complex on an oat culture medium and a mixed substrate for 60 days.
Detailed Description
The invention provides an orchid seed-fungus symbiotic germination complex prepared by the preparation method, which comprises calcium alginate hydrogel and a mixture of orchid seed and mycelial of symbiotic fungus embedded in the calcium alginate hydrogel.
In the present invention, each orchid seed-fungus symbiotic germination complex preferably contains 5-10 orchid seeds, more preferably 6-8 orchid seeds, and most preferably 7 orchid seeds. The mass ratio of the hypha of the symbiotic fungi to the seeds of the orchids is preferably 20 (0.13-0.26), more preferably 20 (0.18-0.22), and most preferably 10: 0.10. the present invention is not particularly limited in kind of the seeds of the orchids, and the seeds of orchids known in the art may be used, for example, those of dendrobe, Bletilla (Bletilla), Paphiopedilum (Paphiopedilum), or Cymbidium (Cymbidium). The species of the dendrobium preferably comprises dendrobium officinale. 0.13g Dendrobium officinale seeds (weight in dry storage state) corresponding to about 25000 seeds. The mass ratio of the calcium alginate hydrogel to the mass of the mixture of the orchid seeds and the mycelia of the symbiotic fungi is preferably 10-15: 20-25, and more preferably 1: 2.
In the invention, the specification of the orchid seed-fungus symbiotic germination complex is as large as pea, the diameter of the complex is 2.8-3.2 mm, the complex is spherical, the complex is transparent and milk white, and the complex has the best seed germination and later seedling growth effects.
The invention provides a preparation method of an orchid seed-fungus symbiotic germination complex, which is characterized by comprising the following steps:
1) preparing hypha of symbiotic fungi into a fungus hypha suspension;
2) mixing the fungal hypha suspension obtained in the step 1) with the orchid seeds to obtain an orchid seed-fungal mixed suspension;
3) mixing the mixed suspension of the seeds of the orchidaceae and the fungi in the step 2) with an aqueous solution of sodium alginate to obtain an orchidaceae seed-fungi embedded body;
4) dripping the orchid seed-fungus embedding body in the step 3) into a calcium chloride aqueous solution for ion exchange reaction to obtain an orchid seed-fungus symbiotic germination complex.
The invention prepares the hypha of the symbiotic fungi into the hypha suspension of the fungi.
In the present invention, the symbiotic fungi include Ceriporioles (Sebacinales), Mycosphaeaceae (Tulasnellaceae) or Ceratopsidae (Ceratobasidiaceae). The fungi of the order Ceriporioidea preferably include Sebacinales LQ, Pilus indica DSM 11827. The Eumycota cerasus preferably comprises Sebacinales LQ strain with the preservation number of CCTCC NO: M2019744. The bacterial strain of Sebacinales LQ is a strain disclosed in the prior art and disclosed in the Chinese patent publication No. CN 110699261A. The family Conidiomycetes (Tulasnellaceae) preferably comprises the genus Mycoplasma (Tulasnella). The family of the carotobasidiomycetes (ceratobasidioceae) preferably comprises the genus carotobasidium (Ceratobasidium).
The method for preparing the fungal hyphae suspension of the present invention is not particularly limited, and a preparation method of the fungal hyphae suspension known in the art may be employed. In the present example, the fungal hyphae were broken up using a homogenizer. The parameters of the refiner breaking are preferably as follows: rated voltage 220v, rated frequency 50Hz, power 350W and revolution 11000-23000 rpm. The time for breaking by a homogenizer is preferably 1.5-2 min. The solvent for the fungal hyphae suspension is preferably a sterile aqueous solution containing 60g/L agar. The hypha concentration of the fungus hypha suspension is 18-22 g/L, and more preferably 20 g/L. The method for producing the fungal hyphae of the present invention is not particularly limited, and a method for culturing fungal hyphae known in the art may be used. In the embodiment of the invention, the Sebacinales LQ strain is preferably subjected to static culture by using a PDA solid culture medium, and then inoculated into a PDB liquid culture medium for shaking culture, and hyphae are separated from the obtained culture solution. The temperature of the static culture is preferably 23-27 ℃, and more preferably 25 ℃. The time for the static culture is preferably 6-8 d, and more preferably 7 d. The rotation speed of the shaking culture is preferably 180-220 rpm, and more preferably 200 rpm. The time of the shaking culture is preferably 9-12 days, and more preferably 10 days. The method for separating hyphae preferably adopts filtration method. The filtration method preferably adopts sterile gauze to filter the obtained culture solution. And washing the hyphae with sterile water to remove residual culture solution, and drying to obtain the fungal hyphae.
After obtaining the fungus hypha suspension, the invention mixes the fungus hypha suspension with the orchid seed to obtain the orchid seed-fungus mixed suspension.
In the invention, the mass ratio of the fungal hyphae to the orchid seeds in the fungal hyphae suspension is preferably 20 (0.13-0.26), more preferably 20 (0.18-0.22), and most preferably 10: 0.10. The density of the orchid seeds in the orchid seed-fungus mixed suspension is 25-50 grains/ml, more preferably 30-45 grains/ml, and even more preferably 35-40 grains/ml. The plant of Orchidaceae includes Dendrobium (Dendrobium), Bletilla (Bletilla), Paphiopedilum (Paphiopedilum) or Cymbidium (Cymbium). The Dendrobium genus preferably includes Dendrobium officinale (Dendrobium officinale Kimura et Migo) the seeds of the Orchidaceae plants are preferably subjected to a sterilization treatment before mixing. The method of sterilization in the present invention is not particularly limited, and a sterilization method known in the art may be used.
After the mixed suspension of the seeds of the orchidaceae plant and the fungi is obtained, the mixed suspension of the seeds of the orchidaceae plant and the fungi is mixed with the sodium alginate aqueous solution to obtain the embedding body of the seeds of the orchidaceae plant and the fungi.
In the invention, the volume ratio of the mixed suspension of the seed-fungus of the orchid family plant to the sodium alginate aqueous solution is preferably (1.8-2.2): 1, and more preferably 2.0: 1. The weight percentage content of the sodium alginate aqueous solution is preferably 2.5-3.5%, and more preferably 3.0%. After mixing, the resulting mixture is preferably thoroughly shaken.
After the orchid seed-fungus embedding body is obtained, the orchid seed-fungus embedding body is dropped into a calcium chloride aqueous solution for ion exchange reaction, and an orchid seed-fungus symbiotic germination complex is obtained.
In the invention, the dosage of the orchid plant seed-fungus embedding body per drop is 9-12 ml, and more preferably 10 ml. The ion exchange reaction is that sodium alginate and calcium chloride generate ion exchange reaction to form calcium alginate hydrogel, seeds and hypha of the orchids can be embedded into the hydrogel to form a closed space, and the germination of the seeds and the growth of later seedlings are promoted under the action of fungi. The concentration of the calcium chloride aqueous solution is preferably 1.5% to 2.5%, most preferably 2.0%. After the ion exchange reaction is completed, it is preferable to perform solidification and washing. The curing time is preferably 15-20 min, and more preferably 18 min. The washing method comprises placing the complex in sterile water, removing after 20min, and absorbing water with filter paper.
In the present invention, the size of the orchid seed-fungus symbiotic germination complexes and the number of orchid seeds contained in each complex can be adjusted according to the seed vitality, the germination promoting effect of different fungi or the practical application conditions, so as to obtain the optimal seed germination rate and seedling rate.
In the invention, the preferable preservation method of the dendrobium officinale seed-fungus symbiotic germination complex is as follows: the prepared dendrobium officinale seed-fungus symbiotic germination complex is put into a sterilized glass bottle, a small amount of sterile water is added, and the dendrobium officinale seed-fungus symbiotic germination complex is sealed and stored in a refrigerator at 4 ℃ in the dark.
The invention provides an application of the orchid seed-fungus symbiotic germination complex in orchid cultivation.
The dendrobium officinale seed-fungus symbiotic germination complex obtained by the invention can be directly applied to seeding under natural conditions and large-scale centralized seedling raising of dendrobium officinale on an artificial mixed matrix. Sowing the orchid seed-fungus symbiotic germination complex for 20 days to detect the seed germination condition, and recording the time of seed germination and protocorm formation; after 60 days of culture, when a large number of seedlings at the early stage of development are produced in the culture dish, all the culture dishes are taken out, and the germination of the seeds and the development of the protocorm are observed and recorded under a stereo microscope. The seed germination and protocorm development were graded according to grading criteria for the germination stage of orchid seeds by Arditti (1967) and other related studies, and the number of seeds or protocorms in each stage was recorded. Seed germination was simplified to 4 stages: non-germination of seeds, germination of seeds (enlargement of the seed embryo and generation of roots), protocorm formation and development (enlargement of the seed embryo breaks through the seed coat to the appearance of the primary meristem), seedling differentiation and development stages (emergence of the first leaf and subsequent growth). It should be noted that this classification standard is a stage artificially divided for research convenience. The statistical result shows that the germination rate of the dendrobium officinale seed-fungus symbiotic germination complex reaches more than 30%, the protocorm rate reaches more than 17%, and the survival rate of seedlings reaches more than 5%.
The orchid seed-fungus symbiotic germination complex provided by the invention and the preparation method and application thereof are described in detail by the following examples, but the invention is not to be construed as being limited by the scope of the invention.
Example 1
Preparation method of symbiotic germination complex of dendrobium officinale seeds and cercospora fungi (Sebacinales LQ)
1. And (3) disinfection of the dendrobium officinale seeds: taking out the seeds from a seed bank stored at-20 ℃, and placing the seeds at room temperature for 10 hours to restore the temperature of the seeds to the room temperature; placing the seeds in a 10ml disposable syringe on a superclean bench, and sealing with non-woven fabrics; sucking 8ml of sodium hypochlorite (NaClO) solution with the concentration of 1% (w/v), shaking to fully contact with the seeds, and sucking sterile water to wash the seeds for 4-5 times after 5min to obtain the sterilized seeds.
2. Preparation of seed-fungus mixed suspension.
2.1 Strain activation: taking out Sebacinales LQ preserved in the inclined plane of a test tube at 4 ℃, inoculating the Sebacinales LQ on a PDA (personal digital assistant) plate culture medium, placing the plate culture medium in an artificial climate box for culture at 25 +/-2 ℃, activating, and taking out the plate culture medium after about 7 days when fungal hyphae grow over the culture dish.
2.2 adding 200ml PDB liquid culture medium into a 300ml conical flask, sealing, filling into a sterilizing pot, and sterilizing at 121 ℃ and 115KPa for 20min for later use; the activated LQ strain was inoculated into a PDB liquid medium to be used, and subjected to fermentation culture at 25 ℃ and 200rpm for 10 days.
And 2.3, filtering the fermentation liquor by using medical sterile gauze to remove the fermentation liquor to obtain conglobate mycelia, washing the conglobate mycelia with sterile water for 4-5 times to remove the residual fermentation liquor in the mycelia, and then sucking the water of the mycelia by using sterile filter paper. Adding 20g of mycelium and 60g of agar into 1000ml of sterile water, putting into a refiner, and crushing, wherein the parameters of the refiner are as follows: the method comprises the following steps of (1) obtaining a fungus suspension of the LQ strain, wherein the rated voltage is 220v, the rated frequency is 50Hz, the power is 350W, the revolution is 11000-23000 rpm, and the breaking time is 1.5min by using a refiner.
2.4 adding the dendrobium officinale seeds subjected to surface sterilization treatment into the fungus suspension of the LQ strain, and uniformly mixing to obtain the dendrobium officinale seed suspension, wherein the added seeds are 0.13g per 1000ml, so that the seed density is controlled to be 25-50 grains/ml, and the dendrobium officinale seed-fungus mixed suspension is obtained.
3. Preparing a dendrobium officinale seed-fungus embedding body: and (3) adding the dendrobium officinale seed-fungus suspension prepared in the step 2.4 into a 3% sodium alginate solution, wherein the volume ratio of the suspension to the embedding solution is 2:1, and fully shaking, mixing and reacting for 8 minutes to obtain the dendrobium officinale seed-fungus embedding body.
4. Solidifying the embedding body to obtain a dendrobium officinale seed-fungus symbiotic germination complex: solidifying the seed-fungus embedded body by adopting a dripping method, sucking the embedded body in the step 3 by using a 10ml disposable syringe, and dripping 10ml to 2 percent of calcium chloride (CaCl) each time2) And (3) carrying out ion exchange reaction in the solution, solidifying for 15 minutes, taking out the formed complex, putting the complex into sterile water, taking out the complex after 20 minutes, sucking water on the surface of the complex by using sterile filter paper, and putting the filter paper in a culture dish paved with the sterile water-impregnated filter paper to obtain the dendrobium officinale seed-fungus symbiotic germination complex.
The prepared complex is in the shape of a round ball with the diameter of about 3.0mm, such as pea size, and is transparent milky white in appearance, and each complex comprises 5-10 seeds of dendrobium officinale (see figure 1).
Example 2
1. Seeding and germination of dendrobium officinale seed-fungus symbiotic germination complex
The dendrobium officinale seed-fungus symbiotic germination complex prepared in example 1 is sown.
1.1 Germination experiments of 2 groups of Dendrobium officinale seed-fungus symbiotic germination complexes are designed, and seeding experiments are carried out on an oat agar medium (OMA) and an aseptic mixed matrix. Subpackaging the sterilized oat agar culture medium into culture dishes, and subpackaging the sterilized mixed medium into disposable lunch boxes.
1.2 sowing the symbiotic germination complex of dendrobium officinale on an oat agar medium (OMA), sowing 20 seeds in each culture dish, respectively setting 22 times of repetition, and sowing 440 seeds in total;
1.3, sowing the dendrobium officinale seed-fungus symbiotic germination complex in a sterilized mixed matrix at the same time, sowing 60 seeds in each lunch box, setting 12 times of sowing for each lunch box, and sowing 720 seeds in total;
the 2 groups of experimental materials above 1.4 are cultured in an artificial incubator at the constant temperature of 25 +/-2 ℃, the illumination intensity is 2500Lx, and the light cycle is 12h/12 h.
2. Seed germination condition detection and data statistical analysis
2.1 detecting the germination condition of the seeds 30 days after sowing, and recording the time of the germination of the seeds and the formation of protocorms; after 60 days of culture, when a large number of seedlings at the early stage of development are produced in the culture dish, all the culture dishes are taken out, and the germination of the seeds and the development of the protocorm are observed and recorded under a stereo microscope. The present example reduces seed germination to 4 stages as a criterion for grading the germination stage of orchid seeds in Arditti (1967) and other related studies: non-germination of seeds, germination of seeds (enlargement of the seed embryo and generation of roots), protocorm formation and development (enlargement of the seed embryo breaks through the seed coat to the appearance of the primary meristem), seedling differentiation and development stages (emergence of the first leaf and subsequent growth). The germination rate (G) of the seeds under each treatment was calculated according to formula I, the protocorm-forming rate (C) under each treatment was calculated according to formula II, and the seedling rate (K) was calculated according to formula III.
G ═ mean (G/t) ± SE formula I
C ═ mean (C/t) ± SE formula II
K ═ mean (K/t) ± SE formula III
Wherein g is the number of germinated seeds in each culture dish under each treatment; c is the number of seeds forming protocorms (the sum of protocorms and seedlings) in each culture dish under each treatment, and k is the number of seedlings in each germination stage in each culture dish under each treatment; t is the number of seeds sowed in each culture dish; SE is standard error.
2.2 in SPSS software (version 25.0) the seed germination rate, protocorm formation rate and seedling rate for different sowing treatments were tested for significance using independent sample t test method, α ═ 0.05. The germination rate, protocorm formation rate and seedling rate of the dendrobium officinale seed-fungus symbiotic germination complex are shown in table 1 after 60 days of symbiotic germination of the dendrobium officinale seed-fungus symbiotic germination complex on different sowing substrates.
TABLE 1 germination of Dendrobium officinale seed-fungus symbiotic germination complex on different substrates for 60 days
Remarking: the statistical data is calculated according to the fact that each dendrobium officinale seed-fungus symbiotic germination complex contains 10 dendrobium officinale seeds on average, and the actual germination rate, protocorm rate and seedling rate are higher than the numerical values in the table.
The results show that the dendrobium officinale seed-fungus symbiotic germination complex has seed germination in any sowing treatment when the symbiotic culture is carried out for 60 days and the experiment is ended (figure 2B). The germination rate, protocorm formation rate and seedling rate of the dendrobium officinale seed-fungus symbiotic germination complex under the two seeding conditions have no obvious difference (all P is more than 0.05) in an oat culture medium and an aseptic mixed medium.
According to the dendrobium officinale seed-fungus symbiotic germination complex prepared by the invention, the dendrobium officinale seed germination can be effectively promoted by embedding the seed-fungus complex, so that protocorms are formed, and the protocorms can be remarkably promoted to develop into seedlings.
Example 3
Preparation method of symbiotic germination complex of dendrobium officinale seeds and pyricularia indica (Serendipita indica DSM11827)
1. And (3) disinfection of the dendrobium officinale seeds: taking out the seeds from a seed bank stored at-20 ℃, and placing the seeds at room temperature for 10 hours to restore the temperature of the seeds to the room temperature; placing the seeds in a 10ml disposable syringe on a superclean bench, and sealing with non-woven fabrics; sucking 8ml of sodium hypochlorite (NaClO) solution with the concentration of 1% (w/v), shaking to fully contact with the seeds, and sucking sterile water to wash the seeds for 4-5 times after 5min to obtain the sterilized seeds.
2. Preparation of seed-fungus mixed suspension.
2.1 Strain activation: taking out the pyrenophora indica (Serendipita indica DSM11827) strain preserved in a test tube inclined plane at 4 ℃, inoculating the strain on a PDA plate culture medium, placing the strain in an artificial climate box for culture at 25 +/-2 ℃, activating, and taking out after about 7 days when fungal hyphae grow over the culture dish.
2.2 adding 200ml PDB liquid culture medium into a 300ml conical flask, sealing, filling into a sterilizing pot, and sterilizing at 121 ℃ and 115KPa for 20min for later use; the activated strain of Pyricularia indica DSM11827 was inoculated into a PDB liquid medium for use, and subjected to fermentation culture at 200rpm at 25 ℃ for 10 days.
And 2.3, filtering the fermentation liquor by using medical sterile gauze to remove the fermentation liquor to obtain conglobate mycelia, washing the conglobate mycelia with sterile water for 4-5 times to remove the residual fermentation liquor in the mycelia, and then sucking the water of the mycelia by using sterile filter paper. Adding 20g of mycelium and 60g of agar into 1000ml of sterile water, putting into a refiner, and crushing, wherein the parameters of the refiner are as follows: rated voltage 220v, rated frequency 50Hz, power 350W, revolution 11000-23000 rpm, breaking time of 1.5min by a refiner, and preparing DSM11827 fungus suspension from broken fungus hyphae.
2.4, adding the dendrobium officinale seeds subjected to surface sterilization treatment into the fungus suspension of DSM11827, and uniformly mixing to obtain a dendrobium officinale seed-fungus suspension, wherein the added seeds are 0.15 g per 1000ml, so as to control the density of the seeds to be 25-50 grains/ml; and adding the seed suspension into a fungus solution of a strain of Pyricularia indica (Serendipita indica DSM11827) to obtain a dendrobium officinale seed-fungus mixed suspension.
3. Preparing a dendrobium officinale seed-fungus embedding body: and (3) adding the dendrobium officinale seed-fungus suspension prepared in the step 2.4 into a 3% sodium alginate solution, wherein the volume ratio of the suspension to the sodium alginate solution is 2:1, and fully shaking, mixing and reacting for 8 minutes to obtain the dendrobium officinale seed-fungus embedded body.
4. Solidifying the embedding body to obtain a dendrobium officinale seed-fungus symbiotic germination complex: solidifying the seed-fungus embedded body by adopting a dripping method, sucking the embedded body in the step 3 by using a 10mL disposable syringe, and dripping 10mL to 2 percent of calcium chloride (CaCl) each time2) And (3) carrying out ion exchange reaction in the solution, solidifying for 15 minutes, taking out the formed complex, putting the complex into sterile water, taking out the complex after 20 minutes, sucking water on the surface of the complex by using sterile filter paper, and putting the filter paper in a culture dish paved with the sterile water-impregnated filter paper to obtain the dendrobium officinale seed-fungus symbiotic germination complex.
5. Seeding and germination of dendrobium officinale seed-fungus symbiotic germination complex
The symbiotic germination complex prepared above and comprising Dendrobium officinale seed and Pyricularia indica DSM11827 fungus was sown.
5.1 Germination experiments of 2 groups of Dendrobium officinale seed-fungus symbiotic germination complexes were designed, and seeding experiments were performed on oat agar medium (OMA) and sterile mixed medium. Subpackaging the sterilized oat agar culture medium into culture dishes, and subpackaging the sterilized mixed medium into disposable lunch boxes.
5.2 sowing the symbiotic germination complex of the dendrobium officinale on an oat agar medium (OMA), sowing 20 seeds in each culture dish, respectively setting 22 times of repetition, and sowing 440 seeds in total;
5.3, sowing the dendrobium officinale seed-fungus symbiotic germination complex in the sterilized mixed matrix at the same time, sowing 60 seeds in each lunch box, setting 12 times of sowing for each lunch box, and sowing 720 seeds in total;
2 groups of experimental materials above 5.4 are cultured in an artificial incubator at the constant temperature of 25 +/-2 ℃, the illumination intensity is 2500Lx, and the light cycle is 12h/12 h.
6. Seed germination condition detection and data statistical analysis
6.1 detecting the germination condition of the seeds 30 days after sowing, and recording the time of the germination of the seeds and the formation of protocorms; after 60 days of culture, when a large number of seedlings at the early stage of development are produced in the culture dish, all the culture dishes are taken out, and the germination of the seeds and the development of the protocorm are observed and recorded under a stereo microscope. The present example reduces seed germination to 4 stages as a criterion for grading the germination stage of orchid seeds in Arditti (1967) and other related studies: non-germination of seeds, germination of seeds (enlargement of the seed embryo and generation of roots), protocorm formation and development (enlargement of the seed embryo breaks through the seed coat to the appearance of the primary meristem), seedling differentiation and development stages (emergence of the first leaf and subsequent growth). The germination rate (G) of the seeds under each treatment was calculated according to formula I, the protocorm-forming rate (C) under each treatment was calculated according to formula II, and the seedling rate (K) was calculated according to formula III.
G ═ mean (G/t) ± SE formula I
C ═ mean (C/t) ± SE formula II
K ═ mean (K/t) ± SE formula III
Wherein g is the number of germinated seeds in each culture dish under each treatment; c is the number of seeds forming protocorms (the sum of protocorms and seedlings) in each culture dish under each treatment, and k is the number of seedlings in each germination stage in each culture dish under each treatment; t is the number of seeds sowed in each culture dish; SE is standard error.
6.2 seed germination rate, protocorm formation rate and seedling rate of different sowing treatments were tested for significance in SPSS software (version 25.0) using independent sample t test method, α ═ 0.05. The germination rate, protocorm formation rate and seedling rate of Dendrobium officinale seed-Pyricularia indica DSM11827 fungus symbiotic germination complex are shown in Table 2 after 60 days of symbiotic germination on different sowing substrates.
TABLE 2 germination of Dendrobium officinale seed-fungus symbiotic germination complexes on different substrates for 60 days
Remarking: the statistical data is calculated according to the fact that each dendrobium officinale seed-fungus symbiotic germination complex contains 10 dendrobium officinale seeds on average, and the actual germination rate, protocorm rate and seedling rate are higher than the numerical values in the table.
The result shows that when the symbiotic culture is carried out for 60 days, the symbiotic germination complex of the dendrobium officinale seed-piriformospora indica DSM11827 and the fungus can germinate well under different sowing conditions. But the germination rate, protocorm rate and seedling rate promoting rate of the complex on the sterile mixed matrix are far higher than those on an oat culture medium, about 2 times more than those on the oat culture medium, and the germination rate of the complex on the sterile mixed matrix is as high as 75.61% +/-7.74%. The dendrobium officinale seed-piriformis indica DSM11827 fungus symbiotic germination complex has obvious difference in germination rate, protocorm forming rate and seedling rate under two sowing conditions in an oat culture medium and a sterile mixed matrix (all P < 0.05).
The invention realizes a new way for symbiotic germination culture of the orchid and the fungus by embedding a novel orchid seed-fungus symbiotic germination complex created by 'seed and fungus', can effectively improve the germination rate of the seed, the growth speed of the seedling and the survival rate of the seedling transplanted to the natural environment, and opens up a new way for large-scale, industrialized and rare frequently dangerous medicinal orchid protection of endangered species, ornamental species, medicinal species and the like of the orchid.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. A preparation method of an orchid seed-fungus symbiotic germination complex is characterized by comprising the following steps:
1) preparing hypha of symbiotic fungi into a fungus hypha suspension;
2) mixing the fungus hypha suspension in the step 1) with the orchid seeds to obtain an orchid seed-fungus mixed suspension;
3) mixing the mixed suspension of the seeds of the orchidaceae and the fungi in the step 2) with an aqueous solution of sodium alginate to obtain an orchidaceae seed-fungi embedded body;
4) dripping the orchid seed-fungus embedding body in the step 3) into a calcium chloride aqueous solution for ion exchange reaction to obtain an orchid seed-fungus symbiotic germination complex.
2. The method of claim 1, wherein the symbiotic fungi comprises the order Ceratophylla (Sebacinales), Mycolepsyllidae (Tulasnellaceae), or Ceratobasidaceae (Ceratobasiidae);
the plant of Orchidaceae includes Dendrobium (Dendrobium), Bletilla (Bletilla), Paphiopedilum (Paphiopedilum), Cymbidium (cymbium) and/or Phyllostachys (Arundina).
3. The method for preparing a orchidaceae plant seed-fungus symbiotic germination complex according to claim 2, wherein the fungus of the order cerifera includes the strains Sebacinales LQ, Pilus piriformis (Serindipita indica DSM 11827);
the plant of the genus Dendrobium includes Dendrobium officinale (Dendrobium officinale Kimura et Migo).
4. The method for preparing an orchid seed-fungus symbiotic germination complex according to claim 2 or 3, wherein the fungus belonging to the order Ceriporioles comprises a Sebacinales LQ strain.
5. The preparation method of the orchid seed-fungus symbiotic germination complex according to claim 1, wherein the mass ratio of fungus hyphae to orchid seeds in the fungus hypha suspension in the step 2) is 20 (0.13-0.26);
the density of the orchid seeds in the orchid seed-fungus mixed suspension in the step 2) is 25-50 grains/ml.
6. The preparation method of the orchid seed-fungus symbiotic germination complex according to claim 1, wherein the volume ratio of the orchid seed-fungus mixed suspension in the step 3) to the sodium alginate aqueous solution is (1.8-2.2): 1;
the mass percentage of the sodium alginate aqueous solution is 2.5-3.5%.
7. The preparation method of the orchid seed-fungus symbiotic germination complex according to claim 1, wherein the orchid seed-fungus inclusion in step 4) is 9-12 ml per drop.
8. The orchid seed-fungus symbiotic germination complex prepared by the preparation method of any one of claims 1 to 7, which is characterized by comprising calcium alginate hydrogel and a mixture of orchid seed and hyphae of symbiotic fungi embedded in the calcium alginate hydrogel.
9. The orchid seed-fungus symbiotic germination complex of claim 8, wherein each orchid seed-fungus symbiotic germination complex comprises 5-10 orchid seeds;
the mass ratio of the hypha of the symbiotic fungi to the seeds of the orchids is 20 (0.13-0.26).
10. Use of an orchid seed-fungus symbiotic germination complex according to claim 8 or 9 in orchid cultivation.
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