CN107018883A - A kind of method for producing banana mycorhiza tissue culture of sprout - Google Patents

A kind of method for producing banana mycorhiza tissue culture of sprout Download PDF

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CN107018883A
CN107018883A CN201710010831.0A CN201710010831A CN107018883A CN 107018883 A CN107018883 A CN 107018883A CN 201710010831 A CN201710010831 A CN 201710010831A CN 107018883 A CN107018883 A CN 107018883A
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culture
lcgx
banana
plant
glomus versiforme
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覃晓娟
陈廷速
李冬萍
张金莲
刘增亮
汪茜
龙艳艳
冯重阳
宋娟
晏卫红
黄京华
陈振松
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Guangxi Wubao Agricultural Technology Co ltd
INSTITUTE OF MICROBIOLOGY GUANGXI ZHUANG AUTONOMOUS ACADEMY OF AGRICULTURAL SCIENCES
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Guangxi Wubao Agricultural Technology Co ltd
INSTITUTE OF MICROBIOLOGY GUANGXI ZHUANG AUTONOMOUS ACADEMY OF AGRICULTURAL SCIENCES
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
    • A01G31/00—Soilless cultivation, e.g. hydroponics
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
    • A01G24/00—Growth substrates; Culture media; Apparatus or methods therefor
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
    • A01H4/001—Culture apparatus for tissue culture
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
    • A01H4/008—Methods for regeneration to complete plants
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor

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Abstract

The invention discloses the application of one plant of Glomus versiforme LCGX 58 and its microbial inoculum in banana planting：(1) stem apex of the banana plant with growing point is subjected to Fiber differentiation, squamous subculture, culture of rootage successively, produces tissue culture seedlings of bananas；(2) training tissue culture seedling in step (1) is taken；(3) transplant, the microbial inoculums of Glomus versiforme LCGX 58 are mixed with seedling medium, cultivate.The microbial inoculum that the present invention is provided has good facilitation to Banana Growth, and adaptive faculty and colonazition are strong, can promote absorption of the banana to nutrient in soil, promote the growth of banana plant；Plant Tissue Breeding, mycorrhizal seedling raising and greenhouse transplantation technique are combined by the present invention, set up the technical system of a set of banana Mycorrhizal tissue culture of sprout nursery, technical support is provided for the factorial praluction of banana, also it is the mycorrhizal seedling raising of other plants simultaneously, quick breeding provides correlation technique foundation.

Description

A kind of method for producing banana mycorhiza tissue culture of sprout
Technical field
The invention belongs to plant Mycorrhizal Technology field, more particularly to banana mycorhiza tissue culture of sprout production technology.
Background technology
Banana is the important industrial crops in south China area, at present, and the two of restriction China banana industry sound development are big Problem is that banana main producing region droop disease is serious and soil fertility is relatively low.Banana blight also known as banana Panama disease, yellow leaf Disease, is a kind of to infect vascular bundle by Cuba specialized form Fusarium oxysporum and cause the wilting crushing fungi soil-borne disease of plant.Clump Branch mycorhiza (AM) fungi is one kind of mycorrhizal fungi, can promote absorption and profit of the plant host to moisture and nutrient (particularly phosphorus) With the great application potential in terms of promoting plant growth, improving soil ecology, suppression soil-borne disease and improve plant disease resistance Microbial resources.
Using Banana Tissue culture technique, large-scale production banana seedling can effectively improve banana seedling quality.Meanwhile, Using Mycorrhizal Technology, the human assistance for carrying out efficient mycorrhizal fungi to tissue-cultured seedling when transplanting is inoculated with, and realizes kind of a seedling mycorrhizal, this Vital effect will be played to improving the transplanting survival rate of Banana Tissue cultivating seedling, improving its upgrowth situation.But at present In the actual production of banana, application of the AM mycorhiza on tissue culture seedlings of bananas is there are no, is not more found a kind of with substantially rush The AM microbial inoculums of raw effect.
The content of the invention
In view of this, the dominant strain of the isolated one plant of AMF of the present invention, Glomus versiforme LCGX-58, AM microbial inoculums are made with the bacterial strain, under the conditions of suitable symbiosis cultivating system, there is significant growth-promoting function to banana.
In order to realize foregoing invention purpose, the present invention provides following technical scheme：
The invention provides one plant of Glomus versiforme (Glomus versiforme) LCGX-58, deposit number is CGMCCNo.8775。
Glomus versiforme (Glomus is being prepared the invention provides Glomus versiforme LCGX-58 described in above-mentioned technical proposal Versiforme) the application in LCGX-58 microbial inoculums.
The invention provides the preparation method of Glomus versiforme LCGX-58 microbial inoculums described in above-mentioned technical proposal, comprising following Step：
(a) using corn as host, by Glomus versiforme LCGX-58 spore inoculating in maize seedling rootses, plantation, training Support；
(b) grow after Glomus versiforme mycelia, the corn in cultivating the step (a) stops watering, and plant to be planted dries up After remove stalk, it is Glomus versiforme LCGX- to retain gained mycelia, culture matrix and maize root system in root system, culture matrix 58 microbial inoculums.
It is preferred that, the corn seedling in step (a) is planted in culture matrix, greenhouse illumination cultivation 12~16 weeks.
It is preferred that, the culture matrix includes river sand and zeolite；
It is preferred that, the volume ratio of the river sand and zeolite is 2:1~3:1；
It is preferred that, the culture matrix is using preceding passing through sterilization treatment.
It is preferred that, LCGX-58 microbial inoculums include gained mycelia, culture matrix and maize root system in culture matrix.
Present invention also offers application of the LCGX-58 microbial inoculums in banana production described in above-mentioned technical proposal, comprising following Step：
(1) stem apex with growing point will be trimmed to after the suction bud aseptic process of banana plant, be in temperature by the stem apex 25 DEG C~28 DEG C, luminous intensity be to carry out induction training successively under the condition of culture that 2000~3000lx, light application time are l2hd-1 35~40 days, squamous subculture 25~30 days and culture of rootage 20~30 days are supported, tissue culture seedlings of bananas is obtained；
(2) tissue culture seedlings of bananas for obtaining the step (1) carries out the hardening of 15~20 days by a definite date；
(3) tissue culture seedlings of bananas after the step (2) hardening is transplanted and cultivated to seedling culture medium, the nursery It is 1 that culture medium, which includes volume ratio,:1~1:2 Glomus versiforme LCGX-58 microbial inoculums and seedling medium.
It is preferred that, maintenance management is carried out in the incubation of the step (3), the maintenance management is specially：Lid plastics Film moisturizing 15~20 days, keeps humidity 70~80%；In the incubation, a Hoagland ' s battalion is sprayed within every 2 weeks Nutrient solution.
It is preferred that, Fiber differentiation inducing culture described in step (1) includes MS basal mediums, accounts for the MS bases BA and account for the NAA that the MS basal mediums concentration is 0.1~0.2mg/L that culture medium concentration is 2~5mg/L.
It is preferred that, squamous subculture subculture medium described in step (1) includes MS basal mediums, accounts for the MS bases BA and account for the NAA that the MS basal mediums concentration is 0.05~0.15mg/L that culture medium concentration is 1~3mg/L.
It is preferred that, culture of rootage root media described in step (1) includes 1/2MS basal mediums, accounts for described 1/ NAA that 2MS basal medium concentration is 0.1~0.2mg/L and to account for the 1/2MS basal mediums concentration be 0.1~0.5% Activated carbon.
Technique effect
The dominant strain of the isolated one plant of AMF of the present invention, Glomus versiforme LCGX-58, with the bacterial strain system Into AM microbial inoculums, gained microbial inoculum has good facilitation to Banana Growth, and adaptive faculty and colonazition are strong, can promote banana to soil The absorption of nutrient in earth, promotes banana plant growth.It is further of the invention by Plant Tissue Breeding, mycorrhizal seedling raising and greenhouse Transplantation technique is combined, and sets up the technical system of a set of banana Mycorrhizal tissue culture of sprout nursery, is factory's metaplasia of banana Production provides technical support, while being also the mycorrhizal seedling raising of other plants, quick breeding provides correlation technique foundation.
Biological deposits explanation
Glomus versiforme (Glomus versiforme) LCGX-58, depositary institution：Chinese microorganism strain preservation is managed Committee's common micro-organisms center, preservation address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Chinese Academy of Sciences microorganism is ground Study carefully institute, preservation date：01 month 2014 16 days, preserving number：CGMCC No.8775.
Brief description of the drawings
Fig. 1 is Glomus versiforme LCGX-58 strain morphology feature；
Fig. 2 is that gained is fragrant after being applied using present invention preparation gained Glomus versiforme LCGX-58 microbial inoculums in banana planting Any of several broadleaf plants mycorhiza plant, wherein AM is the banana mycorhiza plant of addition Glomus versiforme LCGX-58 microbial inoculums, and CK is the sky for being not added with microbial inoculum White control；
Fig. 3 is that Glomus versiforme LCGX-58 microbial inoculums infect detection with control group banana plant root system mycorhiza, and wherein A is bacterium Silk, B is vesicle, and C is clump branch, and D is control group.
Embodiment
The invention provides one plant of Glomus versiforme (Glomus versiforme) LCGX-58, biological deposits numbering is CGMCCNo.8775。
It is preferred that, Glomus versiforme LCGX-58 morphological feature is：Spore：Dan Sheng in soil, it is spherical or subsphaeroidal, it is yellow To yellowish-brown, there is a circle dark along sporoderm under microscope, 82~136 μm of size；
Conidial cell wall：3 layers of sporoderm, L1 water white transparencies easily die wall, about 1 μm, and maturation comes off or only remained part detritus often；L2 is shallow Huang is to yellowish-brown stratiform wall, and thick 2~4 μm, thickness is more uniform；The single walls of L3, are difficult to separate with L2, burnished gold by about 1 μm；
Connect spore mycelia：Water white transparency, it is wide 4.3~6.8 μm, normal atrophy or come off in ripe spore.Even point is wide by 7.6~12.0 μm, straight or small horn, it is open or by an inwall formed every closing；
Inclusion：Oil droplet shape.
Glomus versiforme (Glomus versiforme) LCGX- is being prepared the invention discloses Glomus versiforme LCGX-58 Application in 58 microbial inoculums.
The invention provides the preparation method of the Glomus versiforme LCGX-58 microbial inoculums, comprise the steps of：
(a) using corn as host, by Glomus versiforme LCGX-58 spore inoculating in germination corn seed, plantation, training Support；
(b) grow after Glomus versiforme mycelia, the corn in cultivating the step (a) stops watering, and plant to be planted dries up After remove stalk, it is Glomus versiforme LCGX- to retain gained mycelia, culture matrix and maize root system in root system, culture matrix 58 microbial inoculums.
In the present invention, the germination corn seed that Glomus versiforme spore is vaccinated with root system is planted to enter in culture matrix Row culture.It is preferred that, inoculation method is：Single spore is positioned near germination corn seed or mixed with liquid-transfering gun.
In the present invention, the culture matrix is used for cultivating germination corn seed, preferably includes river sand and zeolite；The river The volume ratio of husky and zeolite is preferably 2:1~3:1, more preferably 3:1.In the present invention, the culture matrix uses preceding preferred By sterilization treatment, the present invention does not have special limitation to the method for the sterilizing, using training well known to those skilled in the art Support matrix sterilization technology scheme；Specifically high pressure steam sterilization can be used in an embodiment of the present invention；Described sterilizing Processing is preferably：The high pressure steam sterilization 1h under the conditions of 121 DEG C, 103.4kPa.
In the present invention, the planting density of the host is preferably 35~45 plants/basin, more preferably 40 plants/basin, described The specification of basin is upper surface diameter × lower surface diameter × a height of 780 × 350 × 250mm of basin.Density is too big, and root system of plant is too close Collection, is unfavorable for infecting for spore；Density is small, and root system is very few, is unfavorable for the growth and breeding of spore.
In the present invention, the culture is illumination cultivation；The illuminance of the illumination cultivation is preferably 2000~3000lx, More preferably 2500lx；The present invention grows earth's surface blastocyst mycelia preferably in illumination cultivation 12~16 weeks, more preferably 13~ 15 weeks, most preferably 14 weeks.It is preferred that, described earth's surface blastocyst mycelia has infected maize root system, reaches commensalism. The temperature of the culture is preferably room temperature, preferably 1~2 day/time of the frequency watered, and irrigation amount, which is preferably up to moistened, does not touch with one's hand i.e. Can.
Grow after Glomus versiforme mycelia, the present invention stops watering to the corn in the culture, removed after plant to be planted is withered Stalk is gone, mycelia, culture matrix and the maize root system retained in root system, obtained culture matrix is Glomus versiforme LCGX- 58 microbial inoculums.
Present invention also offers a kind of Glomus versiforme microbial inoculum, specially preparation method described in above-mentioned technical proposal is obtained Mycelia, culture matrix and maize root system in culture matrix, it is furthermore preferred that being the mixed of the mycelia in culture matrix and culture matrix Compound.The microbial inoculum after testing after directly use, it is preferred that microbial inoculum detect spore concentration, reach 80~120 spores/g.
Present invention also offers application of the LCGX-58 microbial inoculums in banana production described in above-mentioned technical proposal, comprising following Step：
(1) stem apex with growing point will be trimmed to after the suction bud aseptic process of banana plant, be in temperature by the stem apex 25 DEG C~28 DEG C, luminous intensity be to carry out induction training successively under the condition of culture that 2000~3000lx, light application time are l2hd-1 35~40 days, squamous subculture 25~30 days and culture of rootage 20~30 days are supported, tissue culture seedlings of bananas is obtained；
(2) tissue culture seedlings of bananas for obtaining the step (1) carries out the hardening of 15~20 days by a definite date；
(3) tissue culture seedlings of bananas after the step (2) hardening is transplanted and cultivated to seedling culture medium, the nursery It is 1 that culture medium, which includes volume ratio,:1~1:2 Glomus versiforme LCGX-58 microbial inoculums and seedling medium.
The present invention will be specifically trimmed to using stem apex as the raw material of banana culture after the suction bud aseptic process of banana plant Stem apex with growing point.The method of aseptic process is that clear water washes away top layer soil, divests leaf sheath and adventitious root, washing powder is used respectively Water and originally water washing are simultaneously rinsed well, during laboratory operation, bring transfer room into, after being wiped with cotton ball soaked in alcohol, in ultra-clean work On platform, false cauline leaf sheath is peelled off layer by layer again；
Obtain after the stem apex with growing point, the stem apex is carried out Fiber differentiation, squamous subculture and life by the present invention successively Root culture.In the present invention, the Fiber differentiation preferably includes MS basal mediums with inducing culture, accounts for MS bases culture BA and account for the NAA that the MS basal mediums concentration is 0.1~0.2mg/L that base concentration is 2~5mg/L；More preferably include MS Basal medium, to account for BA that the MS basal mediums concentration is 4mg/L and account for the MS basal mediums concentration be 0.15mg/ L NAA.
In the present invention, the temperature of the Fiber differentiation is 25 DEG C~28 DEG C, more preferably 26 DEG C；Intensity of illumination is 2000 ~3000lx, preferably 2500lx；Light application time is l2hd-1.In the present invention, the time of the Fiber differentiation is 35~40 My god, preferably 40 days.
After Fiber differentiation, obtained cultivating seedling is transferred in subculture medium by the present invention carries out squamous subculture.In this hair In bright, the squamous subculture preferably included with subculture medium MS basal mediums, account for the MS basal mediums concentration for 1~ 3mg/L BA and account for the MS basal mediums concentration be 0.05~0.15mg/L NAA；More preferably include the culture of MS bases Base, account for BA and account for the NAA that the MS basal mediums concentration is 0.1mg/L that the MS basal mediums concentration is 2.5mg/L.
In the present invention, the temperature of the squamous subculture is 25 DEG C~28 DEG C；Intensity of illumination is 2000~3000lx, preferably For 2500lx；Light application time is l2hd-1.In the present invention, the time of the squamous subculture is 25~30 days, preferably 30 My god.
After squamous subculture, obtained cultivating seedling is transferred in root media by the present invention carries out culture of rootage.In this hair In bright, the culture of rootage preferably includes 1/2MS basal mediums with root media, to account for the 1/2MS basal mediums dense NAA and account for the activated carbon that the 1/2MS basal mediums concentration is 0.1~0.5% that degree is 0.1~0.2mg/L；More preferably wrap Include 1/2MS basal mediums, account for NAA and account for 1/2MS bases training that the 1/2MS basal mediums concentration is 0.15mg/L Support the activated carbon that base concentration is 0.5%.
In the present invention, the temperature of the culture of rootage is 25 DEG C~28 DEG C；Intensity of illumination is 2000~3000lx, preferably For 2500lx；Light application time is l2hd-1.In the present invention, the time of the culture of rootage is 20~30 days, preferably 30 My god.
Obtain after tissue culture seedlings of bananas, the tissue culture seedlings of bananas is carried out hardening by the present invention.In the present invention, the hardening Time is 15~20 days, preferably 18 days.
After the hardening, the present invention, which transplants the tissue culture seedlings of bananas after obtained hardening to seedling culture medium, carries out nursery Culture, it is 1 that the seedling culture medium, which includes volume ratio,:1~1:2 Glomus versiforme LCGX-58 microbial inoculums and seedling medium.At this In invention, the seedling medium is this area conventional substrate, and main component is turf, vermiculite, perlite, a great number of elements and micro Element.
In the present invention, the nursery incubation is carried out in greenhouse, is supported during the nursery culture Pillar is managed, and the maintenance management is preferably included：Transplant bonnet plastic sheeting moisturizing 15~20 days, keep humidity 70~80%； Spray a Hoagland ' s nutrient solution within every 2 weeks during the nursery culture.In the present invention, the time of the moisturizing is more preferably For 18 days；Humidity in the canopy is more preferably 75%.In the present invention, the Hoagland ' s sprayed during the nursery culture The concentration of nutrient solution is preferably 25~50%, and more preferably 40%；The amount of spraying of the Hoagland ' s nutrient solutions is preferably every Strain 100ml.
Below in conjunction with the embodiment in the present invention, the technical scheme in the present invention is clearly and completely described.It is aobvious So, described embodiment is only a part of embodiment of the invention, rather than whole embodiments.Based on the reality in the present invention Example is applied, the every other embodiment that those of ordinary skill in the art are obtained under the premise of creative work is not made all belongs to In the scope of protection of the invention.
Embodiment 1
Glomus versiforme LCGX-58 screening process is as follows：
(1) rhizosphere soil of the ratoon sugarcane for many years 10g gathered from Guangxi Liucheng County as culture is put into food blending machine In cup, add 600mL deionized waters 5000rpm and centrifuge 3~5s；
(2) step (1) high speed is centrifuged into gained material to pour out, passing sequentially through 3 Soil standard sieves, (aperture is upper strata 0.8mm, middle 0.25mm, lower floor 0.0385mm), most of gravel is remained in food blending machine cup, and every layer is rinsed with flowing water Sieve, until the water of outflow is clear water；
(3) residue in lower floor's sieve is gone in the centrifuge tube that mass concentration is 60% sucrose, 1500 turns/min centrifugations Supernatant is rapid down to aperture 0.0385mm sieves in 3min, centrifuge tube；
(4) rinsed with water and glass culture dish is transferred to after 1~2min of supernatant legacy in sieve, stereomicroscope is seen Examine spore；
(5) color of first observed and recorded spore, size, feature, the cystocarp form for connecting spore mycelia under stereomicroscope Deng；On this basis, it is placed in the fresh AM fungal spores of capillary syring picking on slide, water, lactic acid, lactic acid is added successively After the floating supporting agent such as glycerine, PVLG, in the micro- Microscopic observations of Nikon E-600, the shape of record spore, size, color, surface line Decorations, even spore inclusion, number, width and the shape of spore mycelia, spore ultrastructure, auxiliary cell (autochthonal vesicle), external hyphae And accessory structure produces the features such as sporangium；Aid in, using Melzer's reagents, the blue reagent of cotton, observing the idiosyncrasy of spore simultaneously (whether having Vlei blueness), takes pictures to representative or specific feature；According to the morphological feature of spore, using Sch ü β ler＆Walker (2010) categorizing system, and refering to Schenck＆Perez (1988) " VA mycorrhizal fungis identification handbook and Related web site：http://invam.caf.wvu.edu(INVAM,West Virginia University,USA)；http:// www.zor.zut.edu.pl/Glomeromycota/Taxonomy.html(Department of Plant Pathology, University of Agriculture in Szczecin, Poland) and http://www.lrz.de/~ The schuessler/amphylo/amphylogeny.html materials of identification and the original description for delivering novel species in recent years, enter Retrieval, the identification of row kind.Single cell culture, one are further carried out for doubt kind or possible novel species, New Records Substantial amounts of homologous spore is separately won to obtain, then determines species, another part aids in identification using molecular biology method.AM fungal spores With Melzer's:PVLG=1:1 or PVLG is that slide sample is made in floating supporting agent, and sealing, numbering are simultaneously preserved.
As a result
(1) strain morphology feature
Spore：There is a circle dark along sporoderm under Dan Sheng in soil, spherical or subsphaeroidal, Huang to yellowish-brown, microscope, size 82~136 μm, as shown in Figure 1.
Conidial cell wall：3 layers of sporoderm, L1 water white transparencies easily die wall, about 1 μm, and maturation comes off or only remained part detritus often；L2 is shallow Huang is to yellowish-brown stratiform wall, and thick 2~4 μm, thickness is more uniform；The single walls of L3, are difficult to separate with L2, burnished gold by about 1 μm.
Connect spore mycelia：Water white transparency, it is wide 4.3~6.8 μm, normal atrophy or come off in ripe spore.Even point is wide by 7.6~12.0 μm, straight or small horn, it is open or by an inwall formed every closing.
Inclusion：Oil droplet shape.
(2) qualification result
Comprehensive morphological and molecular biological characteristics, Glomus versiforme are accredited as by bacterial strain.
Embodiment 2
(a) using corn as host, Glomus versiforme LCGX-58 monospore is inoculated near germination corn seed or mixed Close, plant sterilized under through 121 DEG C of high steams 1h, river sand：Zeolite=3:In the river sand zeolite culture matrix of 1 volume ratio, greatly Canopy illumination cultivation 15 weeks, produces Glomus versiforme mycelia；Every 7~10 days casting quality concentration is 25~50% during illumination cultivation Hoagland nutrient solutions 100mL；Wherein, plantation uses potted plant (780 × 350 × 250mm) production model, and host's density is 40 Strain/basin；
(b) corn of culture 12~16 weeks in step (a) is stopped watering, removes stalk after plant to be planted is withered, retain root The maize root system of gained mycelia, culture matrix and reservation is Glomus versiforme LCGX-58 microbial inoculums, gained in system, culture matrix Microbial inoculum miospore amount is except there is the mycelia also spore containing Glomus versiforme in 80~120 spore/g, culture matrix；Inoculation Amount uses 100g Glomus versiforme LCGX-58 microbial inoculums according to every basin (780 × 350 × 250mm) corn.
Embodiment 3
Application of the bacterial strain microbial inoculum in banana production, operating procedure is as follows：
(1) tissue-cultured seedling culture：In the banana region without traditional disease, from growing way stalwartness, the high mother of neat, yield of bearing fruit Strain, takes its complete suction bud just basseted, appearance soil is washed down with clear water, divests the leaf sheath and adventitious root inhaled outside bud, Rinsed well again with running water, 5 × 7cm (diameter × height) size is cut into by bud is inhaled with pocket knife, load sterilized bottle. Wiped and carried out after explant sterilizing with cotton ball soaked in alcohol on super bacterium workbench, each layer vacation cauline leaf sheath is peelled off successively, it is trimmed to 3 × The suction bud of the big small band growing points of 3.5cm (diameter × height), with alcohol (0~1min)+mercuric chloride (12~18min), aseptic water washing 5 Secondary, rip cutting is accessed and entered in inducing culture { MS+BA (4.0mg/L)+NAA (0.15mg/L) } into 2~4 pieces centered on stem apex Subculture training is carried out after row Fiber differentiation, Fiber differentiation 40d in subculture medium { MS+BA (2.5mg/L)+NAA (0.1mg/L) } Support, carry out culture of rootage after squamous subculture 30d in root media { 1/2MS+NAA (0.15mg/L)+activated carbon 0.5% }, Culture of rootage condition is 2000~3000Lx of luminous intensity, light application time l2hd in 25 DEG C~28 DEG C of temperature-1, produce banana group Train seedling；
(2) tissue culture seedlings of bananas hardening：Take the banana tissue culture with 5~6 roots and 3~4 leaves of gained in step (1) Seedling, the hardening of progress 15~20 days by a definite date；
(3) tissue-cultured seedling is after hardening in step (2), by with tissue culture seedlings of bananas more than 6~8 roots, 4~6 leaves Transplanted, by Glomus versiforme LCGX-58 microbial inoculums obtained by above-mentioned preparation, (seedling medium main component is grass with seedling medium Charcoal, vermiculite, perlite and a great number of elements and trace element) by volume 1:1 mixing, tissue culture seedlings of bananas is trained on the culture medium Support；
(4) maintenance management：Lid plastic sheeting moisturizing 15~20 days, keeps humidity after 70~80%, 30 days according to normal group Seedling management is trained, while spraying within every 2 weeks a 50%Hoagland nutrient solution, crop field is planted after cultivating 2~3 months.
Embodiment 4
Banana root system mycorhiza is detected：
Experimental group (AM)：The microbial inoculum that embodiment 2 is obtained is applied in banana planting as described in Example 3；
Control group (CK)：Without microbial inoculum, other operations are identical with experimental group.
Banana seedlings are transplanted to after big Tanaka's plantation 3 months, and 5 young plants are randomly selected from experimental group (AM) and control group (CK) Carry out mycorhiza and infect detection.Banana root system is rinsed well with running water, and filter paper blots water.Root system is cut into the long root segments of 1cm, plus 20%KOH solution is gently rinsed 3 times after being completely soaked root system, 90 DEG C of water-bath 15min with running water, and moisture is drained, and is added Alkaline H2O2The color on dark root system top layer is removed after decolouring 1.5h, while softening root system, decolouring rinses 3-5 after terminating with clear water It is secondary and drain moisture.The glacial acetic acid of volumetric concentration 5% is added, room temperature acidifying 5min outwells glacial acetic acid solution；Add 5% vinegar Sour ink dyes (5mL Parker black writing ink+95mL family expenses light-coloured vinegar), after 66 DEG C of water-bath dyeing 30min, removes dye liquor, uses After clear water is rinsed for several times, being decolourized in clear water, (time is>12h).Decolouring root is broken and is placed on slide, after picking decolorization Root segment lie on slide, 2 root segments of every laid parallel cover 24mm × 50mm cover glasses, with finger by root segment slightly Firmly flatten.Using the micro- sem observations of Olympus BX53-32p02 and taking pictures, and record the upgrowth situation of banana seedlings, such as table 1 It is shown：
Table one：AM groups and control group CK banana seedlings growth indexes are contrasted
Above-mentioned to infect detection to banana plant root system progress AM, as a result as shown in figure 3, A is mycelia in Fig. 3, B is vesicle, C For clump branch, D is control group.As seen from Figure 3, can be observed under microscope the root system of mycorhiza tissue-cultured seedling have obvious mycelia, Clump branch and spore (Fig. 3 A, B, C).
As seen from the above embodiment, in experimental group (AM) tissue culture seedlings of bananas transplant after with after microbial inoculum symbiosis culture 60 days, with The banana seedlings of control group are compared to vigorous growth potential is shown, and plant leaf is dark green, and the number of blade is more, and blade is long, robust plant (figure 2)。
Described above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art For member, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also should It is considered as protection scope of the present invention.

Claims (8)

1. one plant of Glomus versiforme (Glomus versiforme) LCGX-58, deposit number is CGMCC No.8775.
2. the LCGX-58 described in claim 1 is in Glomus versiforme (Glomus versiforme) LCGX-58 microbial inoculums are prepared Application.
3. the preparation method of LCGX-58 microbial inoculums according to claim 2, it is characterised in that comprise the steps of：
(a) using corn as host, by Glomus versiforme LCGX-58 spore inoculating in maize seedling rootses, plantation, culture；
(b) grow after Glomus versiforme mycelia, the corn in cultivating the step (a) stops watering, removed after plant to be planted is withered Stalk is gone, the maize root system for retaining gained mycelia, culture matrix and reservation in root system, culture matrix is Glomus versiforme LCGX-58 microbial inoculums.
4. preparation method according to claim 3, it is characterised in that the corn seedling in the step (a) is planted in training Support in matrix, greenhouse illumination cultivation 12~16 weeks.
5. preparation method according to claim 4, it is characterised in that the culture matrix includes river sand and zeolite, described The volume ratio of river sand and zeolite is 2:1~3:1；The culture matrix is using preceding passing through sterilization treatment.
6. LCGX-58 microbial inoculums prepared by the preparation method described in claim 3~5 any one, it is characterised in that include culture Mycelia, culture matrix and the maize root system of gained in matrix.
7. application of the LCGX-58 microbial inoculums in banana production described in claim 6, is comprised the steps of：
(1) stem apex with growing point will be trimmed to after the suction bud aseptic process of banana plant, by the stem apex temperature be 25 DEG C ~28 DEG C, luminous intensity be that 2000~3000lx, light application time are 10~14hd-1Condition of culture under carry out Fiber differentiation successively 35~40 days, squamous subculture 25~30 days and culture of rootage 20~30 days, obtain tissue culture seedlings of bananas；
(2) tissue culture seedlings of bananas for obtaining the step (1) carries out the hardening of 15~20 days by a definite date；
(3) tissue culture seedlings of bananas after the step (2) hardening is transplanted and cultivated to seedling culture medium, the nursery culture It is 1 that base, which includes volume ratio,:1~1:2 Glomus versiforme LCGX-58 microbial inoculums and seedling medium.
8. application according to claim 7, it is characterised in that carry out maintenance management in the incubation of the step (3), The maintenance management is specially：Lid plastic sheeting moisturizing 15~20 days, keeps humidity 70~80%；In the incubation, Spray within every 2 weeks a Hoagland ' s nutrient solution.
CN201710010831.0A 2017-01-06 2017-01-06 A kind of method for producing banana mycorhiza tissue culture of sprout Pending CN107018883A (en)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102150615A (en) * 2010-12-10 2011-08-17 梁经军 Method for culturing, planting and mycorrhizal production of dendrobium officinale kimura et migo
CN103805524A (en) * 2014-03-05 2014-05-21 广西壮族自治区农业科学院微生物研究所 Glomus versiforme LCGX-58 and applications thereof
CN105408468A (en) * 2013-03-15 2016-03-16 斯波根生物技术公司 Plant growth-promoting bacteria and methods of use
CN106212277A (en) * 2016-07-21 2016-12-14 广西壮族自治区农业科学院微生物研究所 A kind of method producing Rhizoma Zingiberis Recens mycorhiza tissue culture of sprout

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102150615A (en) * 2010-12-10 2011-08-17 梁经军 Method for culturing, planting and mycorrhizal production of dendrobium officinale kimura et migo
CN105408468A (en) * 2013-03-15 2016-03-16 斯波根生物技术公司 Plant growth-promoting bacteria and methods of use
CN103805524A (en) * 2014-03-05 2014-05-21 广西壮族自治区农业科学院微生物研究所 Glomus versiforme LCGX-58 and applications thereof
CN106212277A (en) * 2016-07-21 2016-12-14 广西壮族自治区农业科学院微生物研究所 A kind of method producing Rhizoma Zingiberis Recens mycorhiza tissue culture of sprout

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Application publication date: 20170808