CN103468588B - Tulasnella strain and application thereof in promotion of germination of Dendrobium aphyllum seeds - Google Patents
Tulasnella strain and application thereof in promotion of germination of Dendrobium aphyllum seeds Download PDFInfo
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Abstract
The invention relates to a Tulasnella strain [CGMCC (China General Microbiological Culture Collection Center) No.7552]. According to the invention, the nrDNA ITS (Nuclear Ribonucleic Deoxyribonucleic Acid Internal Transcription Sequence) sequence of the Tulasnella strain is submitted to the database of National Center of Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov/), and the sequence number of the strain is KC796463.1. The Tulasnella strain (CGMCC No.7552) is applied to promotion of germination of Dendrobium aphyllum seeds. The Tulasnella strain forms a symbiotic relationship with the protocorm so as to promote the germination of Dendrobium aphyllum seeds and grow into seedlings. Experiments verify that the strain (CGMCC No.7552) not only can remarkably promote the Dendrobium aphyllum seeds to germinate (under the light condition of 97.92+/-0.51% and under the dark condition of 89.34+/-5.89%), but also can remarkably promote formation of protocorm (under the light condition of 91.82+/-3.03% and under the dark condition of 79.90+9.56%) as well as remarkably promote the protocorm to grow to seedlings subsequently.
Description
Technical field
The invention belongs to biological technical field, relate to one and can form fungal bacterial strain glued membrane bacterium (Tulasnella) bacterial strain (CGMCC No.7552) of symbiotic relationship with the symbiosis of Dendrobium aphyllum (Roxb.) C. E. Fisch． (Dendrobiumaphyllum) protocorm, and promoting the application on Dendrobium aphyllum (Roxb.) C. E. Fisch． seed germination, relate in particular to and a kind ofly can form symbiotic relationship with Dendrobium aphyllum (Roxb.) C. E. Fisch． protocorm and promote its seed germination and grow to effective fungi of plantlet stage.
Background technology
The seed of orchid is very tiny, only has the embryo of ateliosis, and seed germination needs to rely on specific symbiotic effects to obtain nutritive substance under field conditions (factors).The sprouting of Orchid Seeds can be cultivated by non-symbiosis germination and symbiotic germination cultivates two kinds of modes.Although most of orchid is cultivated by non-symbiosis germination, and there is higher germination rate, but the sprigging that this mode obtains is to occurring in nature, poor growth, resisting pathogenic microbes ability, survival rate is lower, causes follow-up growth to be seriously obstructed owing to being difficult to set up symbiotic relationship with the rear fungi contacted simultaneously.Symbiotic germination culture technique refers to sows plant seed and symbiotic effects in specific matrix (substratum) simultaneously, and the method can improve the germination rate of seed, the speed of growth of seedling and the survival rate of seedling after being transplanted to physical environment.Because the symbiotic relationship of Orchid Seeds and fungi has specificity, the symbiotic effects of different Orchid Seeds is different.Determine with Dendrobium aphyllum (Roxb.) C. E. Fisch． Seed Development symbiotic relationship and the effective fungi promoting it to sprout is the key link of cultivating Dendrobium aphyllum (Roxb.) C. E. Fisch． seedling, to be carry out the basis that the primary border of Dendrobium aphyllum (Roxb.) C. E. Fisch． returns work.The seedling obtained by the symbiotic germination of seed and fungi has good environmental compatibility after reverting to natural habitat.Therefore obtaining the effective symbiotic effects promoting seed germination is the first step of carrying out Rare and threatened orchid protection.Utilize seed to move ground symbiotic germination technological guide seed germination and become protocorm, the symbiotic effects be separated in protocorm obtains to promote seed germination effective strain the most efficient method.By Dendrobium aphyllum (Roxb.) C. E. Fisch． seed be separated the fungi obtained carry out symbiotic germination experiment on artificial medium, screening can promote the effective symbiotic effects of Dendrobium aphyllum (Roxb.) C. E. Fisch． seed germination, can be High-efficient Production Mycorrhizal seedling and prerequisite is provided, for the recurrence carrying out Dendrobium aphyllum (Roxb.) C. E. Fisch． lays the foundation.At present, in prior art there are no glued membrane bacterium (Tulasnella) bacterial strain (CGMCC No.7552) and promoting that Dendrobium aphyllum (Roxb.) C. E. Fisch． seed germination grows into the report of purposes in seedling and method.
Summary of the invention
The object of the present invention is to provide a kind of Tulasnella strain, and provide this bacterial strain promoting that Dendrobium aphyllum (Roxb.) C. E. Fisch． seed germination grows into purposes in seedling and method.
In order to realize above-mentioned purpose of the present invention, the invention provides following technical scheme:
A kind of glued membrane bacterium (Tulasnella) bacterial strain (CGMCC No.7552).
The nrDNA ITS sequence of described glued membrane bacterium (Tulasnella) bacterial strain (CGMCC No.7552) submit to US National Biotechnology Information central database (NCBI, http: //
www.ncbi.nlm.nih.gov/), sequence number is KC796463.1.
The biological property of described glued membrane bacterium (Tulasnella) bacterial strain (CGMCC No.7552) is: on PDA flat board, cultivate 7 days its bacterium colonies is light tan, show slightly radial, irregular cycle disperses growth, and aerial hyphae is undeveloped, medium sparse; Optical microphotograph Microscopic observation, mycelia tool barrier film, thick 3.5 – 5.0 μm, most 4.0 μm, the nearly right angle of branch, bifurcation is hung contracting, and distance branch forms barrier film nearby; Old hyphal cell wall has thickening phenomenon; Cultivate and within more than 2 weeks, start to form catenate chlamydospore, chlamydospore quantity is not etc.
Described glued membrane bacterium (Tulasnella) bacterial strain (CGMCC No.7552) is promoting the application on Dendrobium aphyllum (Roxb.) C. E. Fisch． seed germination.
As described in application, glued membrane bacterium (Tulasnella) bacterial strain (CGMCC No.7552), by forming symbiotic relationship with protocorm, promotes that Dendrobium aphyllum (Roxb.) C. E. Fisch． seed germination also grows up to seedling.
As described in application, by by Dendrobium aphyllum (Roxb.) C. E. Fisch． seed and different types of fungi co-cultivation in oat medium, cultivation under optical condition (photoperiod is 12/12h L/D) and dark condition (photoperiod is 0/24hL/D) is being had respectively under 25 ± 2 DEG C of conditions in artificial culture case, make bacterial strain and protocorm form symbiotic relationship, thus promote Dendrobium aphyllum (Roxb.) C. E. Fisch． seed germination and grow up to seedling.
Described glued membrane bacterium (Tulasnella) bacterial strain (CGMCC No.7552) promotes the method for Dendrobium aphyllum (Roxb.) C. E. Fisch． seed germination, glued membrane bacterium (Tulasnella) bacterial strain (CGMCC No.7552) and protocorm form symbiotic relationship, promote Dendrobium aphyllum (Roxb.) C. E. Fisch． seed germination and grow up to seedling.
As described in method, by Dendrobium aphyllum (Roxb.) C. E. Fisch． seed and different types of fungi co-cultivation in oat medium, cultivation under optical condition (photoperiod is 12/12h L/D) and dark condition (photoperiod is 0/24hL/D) is being had respectively under 25 ± 2 DEG C of conditions in artificial culture case, make bacterial strain and protocorm form symbiotic relationship, thus promote Dendrobium aphyllum (Roxb.) C. E. Fisch． seed germination and grow up to seedling.
In order to realize object of the present invention, concrete steps provided by the invention are as follows:
1, bacterial classification of the present invention was deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center on 04 28th, 2013, preserved numbering: CGMCC No.7552.The nrDNA ITS sequence of bacterial strain of the present invention has submitted US National Biotechnology Information central database (NCBI, http://www.ncbi.nlm.nih.gov/) to, and its No. Genbank is: KC796463.1.
2, it is light tan that the glued membrane bacterial strain that the present invention is numbered CGMCC No.7552 cultivates 7 days its bacterium colonies on PDA flat board, and show slightly radial, irregular cycle disperses growth, and aerial hyphae is undeveloped, medium sparse.Optical microphotograph Microscopic observation, mycelia tool barrier film, thick 3.5 – 5.0 μm, most 4.0 μm, the nearly right angle of branch, bifurcation is hung contracting, and distance branch forms barrier film nearby; Old hyphal cell wall has thickening phenomenon; Cultivate and within more than 2 weeks, start to form catenate chlamydospore, chlamydospore quantity is not etc.
3, obtain to Dendrobium aphyllum (Roxb.) C. E. Fisch． seed germination effective symbiotic effects ITS fragment sequence at US National Biotechnology Information central database (NCBI, http://www.ncbi.nlm.nih.gov/) in carry out BLAST compare of analysis, itself and GU166410.1Tulasnellacalospora similarity are up to 99%.Be Tulasnella fungi according to bacterium colony, micro-morphology and molecular biology method by Fungal identification involved in the present invention.
4, Dendrobium aphyllum (Roxb.) C. E. Fisch． seed and Tulasnella strain symbiotic germination are tested
Utilize seed and the fungi symbiotic germination in substratum to test to detect the fungi that is separated to whether have facilitation effect to the seed germination stage and contrast the difference of different fungi to seed germination stage facilitation effect.
The strains tested be stored in 4 DEG C of test tube slants takes out by 4.1, and renewed vaccination, on PDA plate culture medium, is placed in 25 ± 2 DEG C of cultivations in growth cabinet, activates.When hypha,hyphae covers with culture dish soon, (about 7 days) take out as symbiotic germination material.
4.2 preparation symbiotic germination substratum: used medium is oat-agar cultures base (OMA, 4gL
-1oatmeal+8gL
-1agar, pH=5.6 – 5.8).Be poured on by the substratum prepared and revolve in mouth bottle, screw bottle cap, sterilizing (121 DEG C, 20min) is for subsequent use afterwards.
4.3 Seed sterilization: before experiment, the seed be stored in-20 DEG C is taken out, put at room temperature 10 hours, make seed temperature return to room temperature.A small amount of seed is placed in the kind attached bag of papery, with staple, paper bag is sealed.To be immersed in distilled water 5 – 10 minutes by filling seed-bearing paper bag, gently squeeze out bubble.With tweezers paper bag transferred to and fill chlorine bleach liquor's (available chlorine ionic concn is 1%) and containing in the beaker of a washing composition, stir gently or shake beaker.After 10 minutes, paper bag is moved to Bechtop, with aseptic nipper paper bag transferred to and fill in the beaker of sterile distilled water, shake gently.Repeat to rinse seed 3 – 4 times.Squeeze out water unnecessary in paper bag gently, cut off paper bag by sterile scissors.
4.4 sowings and cultivation: the method used according to Dixon (1987) slightly makes an amendment.By aseptic seed and 1gL
-1sterile letheen solution is made into aseptic seed suspension.At the semicircle sterile nylon cloth (aperture is 45 μm) that OMA media surface parallel placement two panels radius is 6cm, draw 150 μ l seed suspension liquid with liquid-transfering gun and evenly sow on every sheet nylon cloth.About 0.5cm is inoculated in the medium
3(1 × 1 × 0.5cm) agar block containing single fungi pure growth, with sealed membrane by culture dish good seal.Be divided into three groups: one group of inoculation from Dendrobium aphyllum (Roxb.) C. E. Fisch． protocorm, be separated the bacterial strain being numbered CGMCC No.7552 obtained; Other 2 groups are set to contrast, wherein inoculate Tulasnella (Tulasnella) fungi (being numbered Fcb4) be separated in sclerophyll orchid (Cymbidium manni) protocorm for one group; Another group does not connect bacterium for blank group (CK).Often organize repetition 14 culture dish, constant temperature culture at 25 ± 2 DEG C in artificial culture case, often organizing each 7 culture dish is having cultivation under optical condition (photoperiod is 12/12h L/D) and dark condition (photoperiod is 0/24h L/D) respectively.
4.5 detect: after planting detect seed germination situation weekly, the time of record seed germination and formation protocorm.After cultivating several weeks, when producing the seedling being in the early development stage in a large number in culture dish, whole culture dish is taken out, observed and recorded seed germination and protocorm developmental state under stereoscopic microscope.Slightly adjust on the grade scale basis of seed germination and protocorm developmental state at Stewart & Zettler (2002), carry out hierarchical statistics to seed germination situation under dark and illumination condition, screening effectively can promote Dendrobium aphyllum (Roxb.) C. E. Fisch． seed germination and can grow to the symbiotic effects of plantlet stage.
In above-mentioned steps, the strain material warehouse-in that step 1 provides three test tube slants to cultivate according to the requirement of culture presevation unit obtains into an accession designation number; The software that the ITS base sequence of order-checking gained is uploaded to Genbank provides simultaneously is also submitted to and is obtained No. Genbank.
Observe bacterial strain micro-morphology described in step 2 and use inserted sheet culture method, mold incubator is cultivated 7 to 10 days under 25 ± 2 DEG C of conditions, gets inserted sheet flaking method film-making routinely.Need according to the observation to select the mycelia of different growing stage carry out observing measure as: be positioned at the mycelial growth time longer visible beading chlamydospore bottom inserted sheet, auxiliary microscope micrometer scale can be measured the thickness of mycelia.
In Molecular Identification described in step 3, adopt CTAB method to extract fungal DNA, pcr amplification the primer is ITS1 and ITS4; PCR reaction system (25 μ l) comprising: 2.5 μ l10 × PCR damping fluids, 0.4 μ l dNTP, 1.5 μ l Mg2+, 1.5 μ l ITS1,1.5 μ l ITS4,0.2 μ l Taq enzyme, 15.4 μ l ddH2O, 2 μ l DNA profilings.Amplified reaction carries out on PCR instrument Perkin Elmer, and following PCR circulation: 94 DEG C of denaturation 3min, circulates 1 time; 94 DEG C of sex change 1min, 51 DEG C of annealing 1min, 72 DEG C extend 1min, 30 circulations; Last 72 DEG C extend 10min.Pcr amplification product is that Shanghai Sheng Gong biotechnology company limited checks order.To the row that check order submit to US National Biotechnology Information central database to compare, tentatively confirm the lower status of its classification.
The suspension of 150 microlitres described in step (4.4) is about containing 150 Dendrobium aphyllum (Roxb.) C. E. Fisch． seeds, and described culture condition is: intensity of illumination is 2000 – 3000Lx, temperature 25 ± 2 DEG C, photoperiod 12h/12h L/D.
Cultivation described in step (4.5) will be determined according to seed germination situation several weeks, selects for 8 to 10 weeks, because seed major part has been sprouted and some reaches plantlet stage in this invention; Described seed germination and protocorm developmental state carry out classification with reference to the method for Stewart & Zettler (2002) to seed germination and protocorm developmental state, be divided into 5 stages: it is transparent that the stage 0 is described as embryo, seed coat is intact, and seed is not sprouted; Stage 1 is described as embryo water-swelling; Stage 2 is described as embryo and continues to expand, and breaks through seed coat, is considered as sprouting; Stage 3 is described as occurring protomeristem, namely develops into protocorm; Stage 4 is described as growing first blade; Stage 5 is described as the continued growth of first blade, elongated.
Reference is: Dixon is orchid growing for pleasure and profit K.1987.Modern, orchid club ofsouth Australia:Inc., Adelaide.;
Stewart SL,Zettler LW.2002.Symbiotic germination of three semi-aquatic rein orchids(Habenaria repens,H-quinquiseta,H-macroceratitis)from Florida.Aquatic Botany72:25-35.。
Compared with prior art, the present invention possesses following remarkable excellent benefit:
The sprouting of Orchid Seeds can be cultivated by non-symbiosis germination and symbiotic germination cultivates two kinds of modes.Although most of orchid is cultivated by non-symbiosis germination, and there is higher germination rate, but the sprigging that this mode obtains is to occurring in nature, poor growth, resisting pathogenic microbes ability, survival rate is lower, causes follow-up growth to be seriously obstructed owing to being difficult to set up symbiotic relationship with the rear fungi contacted simultaneously.Symbiotic germination culture technique refers to sows plant seed and symbiotic effects in specific matrix (substratum) simultaneously, and the method can improve the germination rate of seed, the speed of growth of seedling and the survival rate of seedling after being transplanted to physical environment.Because the symbiotic relationship of Orchid Seeds and fungi has specificity, the symbiotic effects of different Orchid Seeds is different.Determine with Dendrobium aphyllum (Roxb.) C. E. Fisch． Seed Development symbiotic relationship and the effective fungi promoting it to sprout is the key link of cultivating Dendrobium aphyllum (Roxb.) C. E. Fisch． seedling, to be carry out the basis that the primary border of Dendrobium aphyllum (Roxb.) C. E. Fisch． returns work.Dendrobium aphyllum (Roxb.) C. E. Fisch． seed and different fungies and contrast are cultivated by symbiotic germination experiment by bacterial strain of the present invention respectively on oat medium, by the effective strain relatively successfully obtaining promotion Dendrobium aphyllum (Roxb.) C. E. Fisch． seed germination of seed germination rate, thus open up a new way for utilizing Dendrobium aphyllum (Roxb.) C. E. Fisch． seed and mycosymbiosis to sprout high effect culture seedling.
The CGMCC No.7552 bacterial strain that the present invention is separated to from the protocorm that original place symbiotic germination produces, seed by Dendrobium aphyllum (Roxb.) C. E. Fisch． puts into special seed packet, seed packet is placed on the Proterozoic of Dendrobium aphyllum (Roxb.) C. E. Fisch． plant strain growth, induction seed germination produces protocorm, artificial culture medium culturing is aseptically used by after the protocorm surface sterilization of acquisition, endogenetic fungus growth in induction protocorm, in time growing mycelia, carry out the purifying of fungi, obtain pure bacterium colony, and carry out Molecular Identification and the preservation of fungi.This bacterial strain significantly can not only promote that Dendrobium aphyllum (Roxb.) C. E. Fisch． seed germination is (under illumination condition 97.92 ± 0.51%; Under dark condition 89.34 ± 5.89%), and significantly can promote that the formation of protocorm is (under illumination condition 91.82 ± 3.03%; Under dark condition 79.90 ± 9.56%) and significantly promote that protocorm subsequent development becomes seedling.Explanation only has inoculating strain CGMCC No.7552 could effectively promote under illumination condition, and Dendrobium aphyllum (Roxb.) C. E. Fisch． seed growth grows plantlet stage.
Embodiment
In order to make object of the present invention, technical scheme and advantage clearly understand, by the following examples and testing data, the present invention is described in more detail.Should be appreciated that specific embodiment described herein only in order to explain the present invention, be not intended to limit the present invention.
Embodiment 1:
Utilize seed and the fungi symbiotic germination in substratum to test to detect the fungi that is separated to whether have facilitation effect to the seed germination stage and contrast the difference of different fungi to seed germination stage facilitation effect:
1, first obtain Tulasnella strain (preserve numbering: CGMCC No.7552):
The seed of Dendrobium aphyllum (Roxb.) C. E. Fisch． is put into seed packet, seed packet is placed on the Proterozoic of Dendrobium aphyllum (Roxb.) C. E. Fisch． plant strain growth, induction seed germination produces protocorm, artificial culture medium culturing is aseptically used by after the protocorm surface sterilization of acquisition, endogenetic fungus growth in induction protocorm, in time growing mycelia, carry out the purifying of fungi, obtain pure bacterium colony.
By by Dendrobium aphyllum (Roxb.) C. E. Fisch． seed and different types of fungi co-cultivation in oat medium, in artificial culture case, having cultivation under optical condition (photoperiod is 12/12h L/D) and dark condition (photoperiod is 0/24h L/D) respectively under 25 ± 2 DEG C of conditions, result shows this bacterial strain and can form symbiotic relationship with protocorm and significantly promote Dendrobium aphyllum (Roxb.) C. E. Fisch． seed germination and grow up to seedling; Determine the taxonomy of this fungi by colony characteristics, micromorphology characteristic sum molecular biology method simultaneously and be stored in China Committee for Culture Collection of Microorganisms's common micro-organisms center.
The determination of fungal taxonomy status:
Morphological specificity is observed: it is light tan that the glued membrane bacterial strain of the above-mentioned CGMCC of being numbered No.7552 cultivates 7 days its bacterium colonies on PDA flat board, and show slightly radial, irregular cycle disperses growth, and aerial hyphae is undeveloped, medium sparse.Opticmicroscope feature: mycelia tool barrier film, thick 3.5 – 5.0 μm, most 4.0 μm, the nearly right angle of branch, bifurcation is hung contracting, and distance branch forms barrier film nearby; Old hyphal cell wall has thickening phenomenon; Cultivate and within more than 2 weeks, start to form catenate chlamydospore, chlamydospore quantity is not etc.
Molecular biology identification: take out the fungal bacterial strain preserved, with in liquid potato glucose (PDB) substratum after aseptic inoculation pin picking mycelia access sterilizing on Bechtop.Put into the concussion of concussion shaking table 25 ± 2 DEG C to cultivate.Incubation time, depending on the speed of growth of fungi, is generally 3 – 6 days.Suction filtration is about the mycelium of 100mg, adopts conventional CTAB method to extract DNA, selects ITS1 and ITS4 to be that primer carries out pcr amplification reaction and carries out agarose gel electrophoresis detection to amplified production.The sample detected containing target fragment is checked order.Submit the ITS fragment sequence obtained to US National Biotechnology Information central database (NCBI, http://www.ncbi.nlm.nih.gov/), and carry out BLAST comparative analysis, obtaining No. GenBank is: KC796463.1.The ITS sequence obtained and accession number are that the fungi (Tulasnellacalospora) of GU166410.1 is the most similar, and maximum similarity reaches 99%, and this identification of strains is Tulasnella fungi by combining form credit category feature.This bacterial classification has adopted test tube slant method to be deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center on 04 28th, 2013, preserves numbering: CGMCC No.7552, storage life: 30 years.Simultaneously the applicant in laboratory by inoculation in some test tube slants, cultivating 14d in 25 ± 2 DEG C of mold incubators, to be placed on 4 DEG C of Storage in refrigerator for subsequent use.
2, symbiotic germination substratum
Used medium is oat-agar cultures base (OMA, 4gL
-1oatmeal+8gL
-1agar, PH=5.6 – 5.8).Be poured on by the substratum prepared and revolve in mouth bottle, screw bottle cap, sterilizing (121 DEG C, 20min) is for subsequent use afterwards.
3, strains tested is heavily cultivated
Strains tested step 1 be stored in 4 DEG C of test tube slants takes out, and renewed vaccination, on PDA substratum, is placed in 25 ± 2 DEG C of cultivations in growth cabinet, activates.Take out as symbiotic germination material when hypha,hyphae covers with culture dish soon.
4, Seed sterilization
Before experiment, the seed be stored in-20 DEG C is taken out, put at room temperature 10 hours, make seed temperature return to room temperature.A small amount of seed is placed in the kind attached bag of papery, with staple, paper bag is sealed.To be immersed in distilled water 5 – 10 minutes by filling seed-bearing paper bag, gently squeeze out bubble.With tweezers paper bag transferred in the beaker filling chlorine bleach liquor's (available chlorine ionic concn is 1%) and a washing composition, stir gently or shake beaker.After 10 minutes, paper bag is moved to Bechtop, with aseptic nipper paper bag transferred to and fill in the beaker of sterile distilled water, shake gently.Repeat to rinse seed 3 – 4 times.Squeeze out water unnecessary in paper bag gently, cut off paper bag by sterile scissors and obtain sterilizing seed.
5, sowing and cultivation
The method that Dixon (1987) is used slightly makes an amendment.By aseptic seed and 1gL
-1sterile letheen solution is made into aseptic seed suspension.At the semicircle sterile nylon cloth (aperture is 45 μm) that OMA media surface parallel placement two panels radius is 6 ㎝, draw 150 μ l seed suspension liquid (about containing 150 seeds) with liquid-transfering gun and evenly sow on every sheet nylon cloth.About 0.5cm is inoculated in the medium
3(1 × 1 × 0.5cm) agar block containing single fungi pure growth, with sealed membrane by culture dish good seal.Inoculate the bacterial strain being numbered CGMCC No.7552 be separated in Dendrobium aphyllum (Roxb.) C. E. Fisch． protocorm for one group, one group of inoculation is separated the bacterial strain (Tulasnella sp.) being numbered Fcb4 obtained from the blue protocorm of sclerophyll, and control group (CK) is not for connect bacterium.Often organize repetition 14 culture dish, constant temperature culture at 25 ± 2 DEG C in artificial culture case, often organizing each 7 culture dish is having cultivation under optical condition (photoperiod is 12/12h L/D) and dark condition (photoperiod is 0/24h L/D) respectively.Be placed in growth cabinet with sealed membrane sealing and cultivate.Culture condition is: intensity of illumination is 2000 – 3000Lx, temperature 25 ± 2 DEG C, photoperiod 12h/12h L/D.
6, detect
After planting detect seed germination situation weekly, the time of record seed germination and formation protocorm.When there being in culture dish the seedling producing and be in the early development stage in a large number, whole culture dish being taken out after cultivating several weeks, examining under a microscope record seed germination and protocorm developmental state.Slightly adjust on the grade scale basis of seed germination and protocorm developmental state at Stewart & Zettler (2002), classification carried out to seed germination situation, adds up the seed germination rate (G) under each process, form protocorm ratio (C) and be in the ratio (K) of the seed in each sprouting stage, protocorm or seedling.G=mean(g/t)±SE,C=mean(c/t)±SE，K=mean(k/t)±SE。Wherein, g is the seed number sprouted in each culture dish under often kind of process; C is the seed number (stage 2,3,4 sum) forming protocorm in the lower each culture dish of often kind of process, k be often kind process under each culture dish in be in the quantity of the seed in each sprouting stage, protocorm or seedling; T is the seed number sowed in each culture dish; SE is standard error.
In R software (version2.14.2), utilize nonparameter test method Kruskal-Wallis test (KW) and Mann-Whitney U – test (U) to the seed germination rate of different treatment, form protocorm ratio and each stage seed number ratio carries out test of significance, α=0.05.
Result does not all have seed germination under showing not connect the illumination of bacterium control group and dark culturing condition, and a seed water-swelling reaches the stage 1 and do not form protocorm; And the CGMCC No.7552 bacterial strain that is separated to significantly can not only promote that Dendrobium aphyllum (Roxb.) C. E. Fisch． seed germination is (under illumination condition 97.92 ± 0.51% from the protocorm that original place symbiotic germination produces; Under dark condition 89.34 ± 5.89%), and significantly can promote that the formation of protocorm is (under illumination condition 91.82 ± 3.03%; Under dark condition 79.90 ± 9.56%) and significantly promote that protocorm subsequent development becomes seedling.Although FCb4 bacterial strain significantly to promote under illumination condition that seed germination forms protocorm (43.79 ± 20.45%) having, show cessation of growth cessation after forming protocorm, continued growth cannot develop into seedling.Explanation only has inoculating strain CGMCC No.7552 could effectively promote under illumination condition, and Dendrobium aphyllum (Roxb.) C. E. Fisch． seed growth grows plantlet stage.
Claims (2)
1. a glued membrane bacterium (
tulasnella sp.) bacterial strain, it is characterized in that, this bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and deposit number is: CGMCC No.7552.
2. glued membrane bacterium according to claim 1 (
tulasnella sp.) application of bacterial strain on promotion Dendrobium aphyllum (Roxb.) C. E. Fisch． seed symbiotic germination.
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| CN105519416B (en) * | 2015-12-28 | 2018-01-19 | 中国科学院西双版纳热带植物园 | A kind of artificial breeding method of dendrobium devonianum symbiosis seedling |
| CN106069652B (en) * | 2016-02-26 | 2019-03-05 | 中国科学院西双版纳热带植物园 | A method of it directly sowing dendrobium devonianum seeds on tea tree and cultivates seedling |
| CN106386495A (en) * | 2016-10-09 | 2017-02-15 | 上海兰葹生物科技有限公司 | Novel Herba Dendrobii culturing method |
| CN110881478B (en) * | 2019-11-07 | 2021-04-13 | 中国林业科学研究院林业研究所 | Method for promoting germination of seeds of denatolum and paphiopedilum by using ceriferous fungi |
| CN114395485B (en) * | 2022-01-11 | 2023-06-23 | 云南大学 | Adhesive film fungus strain TP-2 capable of promoting stem thickness of dendrobium nobile and application |
| CN114381379B (en) * | 2022-01-11 | 2023-06-09 | 云南大学 | Adhesive film fungus strain TP-8 with dendrobium seedling tillering improving capability and application thereof |
| CN114395486B (en) * | 2022-01-11 | 2023-06-09 | 云南大学 | Adhesive film fungus strain TP-3 with high growth promoting capability of dendrobium and application thereof |
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