CN102154115B - Method and special bacterial strain for cultivating arethusa plant - Google Patents
Method and special bacterial strain for cultivating arethusa plant Download PDFInfo
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Abstract
The invention provides a method and special bacterial strain for cultivating an arethusa plant. The bacterial strain provided by the invention is Mycosphaerella sp. DX08 CGMCC No.4149. The method is suitable for sprout cultivation of various arethusa plants; and by using the method, the stress resistance of a seedling can be improved, the cultivation period of the plant seedling can be shortened, and the production cost is reduced. The method has the characteristics that the operation is simple, the cost is low and the like. Thus, the method is suitable for being applied to the scale cultivation of high-quality seedlings, and has important application and popularization value in the aspects of gardening and agriculture.
Description
Technical field
The present invention relates to a kind of method and special strain therefore of cultivating orchid.
Background technology
Orchid has 800 to belong to kind more than 25000 approximately, is distributed widely in the torrid zone, subtropics and Temperate Region in China.China has 180 to belong to 1500 kinds approximately, mainly is distributed in the most areas on the south the Qinling Mountains, and is especially in the majority with Yunnan, Sichuan, Hainan, Taiwan, Guangdong, Guangxi.Orchid is a special phyto-group, is one of large section that evolves the most in the angiosperm.Wherein, the bletilla striata, rhizoma Gastrodiae, the stem of noble dendrobium etc. can be used as medicine; Pocket orchid, lady's slipper etc. have ornamental value.Therefore, along with the demand in market, the destruction of wild resource, people begin large-area artificial culture orchid.
Orchid is typical mycorrhizal plants, has formed to rely on the characteristic that mycorhiza survives and grows in long-term evolutionary process.Many orchids particularly saprophytic orchid have produced absolute dependency to mycorhiza, do not have under field conditions (factors) mycorhiza with regard to poor growth, even dead.All cymbidium seeds all need could sprout with mycosymbiosis under field conditions (factors), and forming mycorhiza is the prerequisite that they survive under field conditions (factors) and grow.Existing result of study shows, the symbiosis fungi helps to improve surviving rate and the speed of growth behind the seedling replanting, increases plant to the resistivity of adverse circumstance, keeps lower sickness rate, increases the quantity of flower and improves the quality etc. of flower.
The seed of orchid is very little, just has several ten thousand in the capsule to up to a million seeds, is dust-like.At present, obtain the main path that seedling is cultivation by the technology of tissue culture.And the aseptic seeding technology of seed is the effective way of obtaining the mass seedling.But traditional aseptically sowing seeds technology is small and light because of Orchid Seeds, and nutrition is deficient, do not have mycosymbiosis and cause that germination rate is low, the impact such as growth of seedling potential difference, resistance are weak.
Summary of the invention
An object of the present invention is to provide the bacterium that a strain is used for cultivating orchid.
Bacterium provided by the present invention is ball chamber bacterium (Mycosphaerella sp.) DX08, and its deposit number is CGMCCNO.4149.
The application of ball chamber bacterium (Mycosphaerella sp.) DX08CGMCC No.4149 in cultivating orchid also belongs to protection scope of the present invention.
In the above-mentioned application, described orchid is Dendrobium Sw; Described Dendrobium Sw is Herba Dendrobii (Dendrobium officinale).
Another object of the present invention provides a kind of method of cultivating orchid.
The method of cultivation orchid provided by the present invention comprises the steps: Orchid Seeds and ball chamber bacterium (Mycosphaerella sp.) DX08CGMCC No.4149 are cultivated altogether, obtains the orchid seedling.
In the aforesaid method, described method of cultivating altogether comprises the steps: described Orchid Seeds is seeded in the substratum, place again the cultivation bacterium piece of ball chamber bacterium (Mycosphaerella sp.) DX08CGMCC No.4149 to media surface after planting, cultivate again.
In above-mentioned arbitrary described method, the amount of described sowing is every 5cm
2Substratum in the sowing 100-200 or 100 or 150 or 200 described Orchid Seeds; The amount of the cultivation bacterium piece of described placement ball chamber bacterium (Mycosphaerella sp.) DX08CGMCC No.4149 is every 5cm
2Media surface place that the 1-2 block is long-pending to be 2mm
3Described cultivation bacterium piece.
In above-mentioned arbitrary described method, described substratum is oat medium;
1 liter of described oat medium is comprised of 2.5g oatmeal, 8g agar and water, and water is supplied volume; The pH value of described oat medium is 5.8;
In above-mentioned arbitrary described method, the cultivation bacterium piece of described ball chamber bacterium (Mycosphaerella sp.) DX08CGMCC No.4149 is to obtain according to the method that comprises the steps: ball chamber bacterium (Mycosphaerella sp..) DX08CGMCC No.4149 is inoculated in the fungi culture medium, 25 ℃ of lower dark cultivations 10 days-20 days or 10 days or 15 days or 20 days, get the bacterium piece from the bacterium colony center.
In above-mentioned arbitrary described method, described temperature of cultivating altogether is 22 ℃-24 ℃ or 22 ℃ or 23 ℃ or 24 ℃, the described periodicity of illumination of cultivating altogether is that 14h illumination/10h is dark, and described intensity of illumination of cultivating altogether is 2000Lx～3000Lx or 2000Lx or 2500Lx or 3000Lx.
In above-mentioned arbitrary described method, described fungi culture medium is the DE substratum.
In above-mentioned arbitrary described method, the described time of cultivating altogether was 5 weeks.
In above-mentioned arbitrary described method, described orchid is Dendrobium Sw.
In above-mentioned arbitrary described method, described Dendrobium Sw is Herba Dendrobii (Dendrobium officinale).
The inventive method has overcome that plant seed is little because of volume, under-nutrition causes the low difficulty of germination rate, has realized the symbiotic germination of dust-like plant seed and fungi, has broken away from the constraint of traditional seed aseptic seeding growing seedlings.Experimental result shows that the method that plant seed of the present invention and mycosymbiosis are sprouted is effective, and the seed germination success ratio is high.The inventive method is applicable to multiple orchid seed cultivation, not only can improve the resistance of seedling, and can shorten the cultivation period of plant seedlings, reduces production costs.The inventive method is the characteristics such as simple to operate, with low cost in addition.Therefore, the inventive method is fit to be applied to the propagation in scale of high quality seedling, has important application valency value at gardening and agricultural.Utilizing the mycorrhizal fungi symbiotic characteristic of orchid, obtain Orchid Seeds and mycosymbiosis and sprout, obtain the Mycorrhizal seedling of mass, is the important channel of effective breakthrough orchid cultivation bottleneck.
Description of drawings
Fig. 1 is the form of bacterium.
Fig. 2 is the tissue cultured seedling form of Herba Dendrobii.
Embodiment
Employed experimental technique is ordinary method if no special instructions among the following embodiment.
Used material, reagent etc. if no special instructions, all can obtain from commercial channels among the following embodiment.
The seed that uses among the following embodiment is the seed of Herba Dendrobii (Dendrobium officinale Kimura et Mi go), document " He Pingrong, Song Xiqiang, Luo Yibo; He Minggao, [in the "Danxia" landform habitat reproductive biology of iron sheet stone solution research (J)]; CHINA JOURNAL OF CHINESE MATERIA MEDICA, 2009,34 (2); 124-127 " in disclosed, the public can obtain from University Of Hainan.
The separation of embodiment 1, bacterial strain and evaluation
One, separation method
Isolate bacterial strain from the wild Herba Dendrobii (Dendrobium officinale Kimura et Migo) that picks up from the Xinning, Hunan, its method is as follows:
1.1, choose the fresh feeding root of the healthy and strong and eugonic wild magnificent stem of noble dendrobium of wild growing way, flowing water rinses out root surface soil ulmin and foreign material.
1.2, on super bacterium worktable, root segment immersed that 10-12min carries out surface sterilization in 1% calcium hypochlorite solution, then use aseptic water washing 3-4 time.
1.3, root segment is cut into the long segment of 2-3mm, be placed on the PDA substratum, 25 ℃ of lower dark cultivations, after cultivating after a while, until mycelia grew up to bacterium colony in root segment after, the picking mycelia was transferred to and carries out culture purified on the PDA substratum.
1.4, do not pollute, change PDA slant medium in the finger type pipe over to, 4 ℃ of short-term preservations are stand-by.
Every liter of PDA substratum is by 200 gram peeled potatoes, 20g sucrose, and 20g agar powder and 1L water are made, the PH nature.Bacterial strain and target seedling that separation is obtained carry out respectively symbiosis culture, detect this bacterial strain behind the certain hour to the size (detecting following index: fresh weight, plant height, joint number, the number of blade of seedling increase in the unit time) of the promoter action of the growth of seedling, each index all is higher than control group, determines that then this bacterium is beneficial bacterium.
The result obtains a strain bacterium, called after DX08.
Two, identify
Bacterial strain DX08 observes the form of bacterium on the PDA substratum.The result: its mycelia is that ascostroma life under host's blade table cortex; [Dan is for burying life, sphere, or oblate, and the aperture is flat or be papilla. and thecaspore is oval, and is colourless, two born of the same parents' equal and opposite in directions (Fig. 1, A are visual inspection, and B is microscopic examination).
Detect the ITS rDNA gene in this bacterial strain, the sequence that obtains is shown in SEQ ID NO:1.This gene order is carried out the blast comparison in the ncbi database.Comparison result shows that this sequence and mycosphaerella (Mycosphaerella sp.) similarity is 100%.
Comprehensive above qualification result shows that this bacterial strain belongs to ball chamber bacterium (Mycosphaerella sp.).This bacterial strain is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on September 13rd, 2010 and (is called for short CGMCC, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101), preserving number is CGMCC No.4149, and Classification And Nomenclature is ball chamber bacterium (Mycosphaerella sp.).
Embodiment 2, cultivation Herba Dendrobii
One, the pre-treatment of Seeds of Dendrobium Candidum
With to grow Herba Dendrobii good ripe and cracking fruit pod put into 1.5% (quality percentage composition) Losantin aqueous solution 13-15min and carry out surface sterilization, aseptic water washing 3-4 time.
Two, the preparation of bacterium piece
1, activation
Ball chamber bacterium (Mycosphaerella sp.) DX08CGMCC No.4149 is inoculated in the PDA substratum, 25 ℃ of lower dark activation of cultivating.
The preparation of PDA substratum: with identical in the prior art, every liter of PDA substratum restrains peeled potatoes by 200,20g sucrose, and 20g agar powder and 1L water are made, the PH nature.
2, the bacterium piece is cultivated
To be inoculated in the culture block of single pure mycelia in the DE substratum, 25 ℃ of lower dark cultivations 15 days, cut the bacterium piece from the bacterium colony center, for subsequent use.
The DE substratum forms: be substratum commonly used in the prior art.
Each rises substratum specifically is the CaCl of 110.98mg/L by final concentration
2, final concentration is the K of 174.25mg/L
2SO
4, final concentration is the MgSO of 123.24mg/L
4.7H
2O, final concentration are the KH of 54.44mg/L
2PO
4, final concentration is the H of 1.55mg/L
3BO
4, final concentration is the MnCl.4H of 6.53mg/L
2O, final concentration are the ZnSO of 0.80mg/L
4.7H
2O, final concentration are the NaMO of 0.24mg/L
4.2H
2O, final concentration are the C of 52.11mg/L
10H
14N
2O
8Na
2.2H
2O, final concentration are the FeSO of 27.80mg/L
4.7H
2O, final concentration are the Zulkovsky starch of 9g/L, the yeast extract paste that final concentration is 1g/L, agar and the water composition that final concentration is 8g/L, and its final pH value is 6.0.
Zulkovsky starch is available from Chemical Reagent Co., Ltd., Sinopharm Group, and catalog number is 10021318.Yeast extract paste is available from Chemical Reagent Co., Ltd., Sinopharm Group, and catalog number is 69024838.
Three, cultivate altogether
Seeds of Dendrobium Candidum after 150 sterilizations is seeded in 5cm
2In the oat medium, place the long-pending 2mm of being of 2 blocks to media surface after planting simultaneously
3The bacterium piece of ball chamber bacterium (Mycosphaerella so.) DX08CGMCC No.4149; Be that 23 ℃, periodicity of illumination are that 14h illumination/10h dark and intensity of illumination are to cultivate for 5 weeks under the condition of 2500Lx in temperature, obtain Herba Dendrobii seedling (Fig. 2).(among Fig. 2, A: the bacterium symbiosis culture is arranged, B: aseptic symbiosis culture).
The composition of oat medium: be comprised of oatmeal, agar and water, the concentration of oatmeal in substratum is 2.5g/L, and the concentration of agar in substratum is 8g/L, and water is supplied volume; The pH value of described oat medium is 5.8.
Contrast: except the bacterium piece that does not access ball chamber bacterium (Mycosphaerella sp.) DX08CGMCC No.4149, all the other steps are all identical with experimental group.
3 repetitions are established in experiment, and the result takes the mean.
Four. the result
1), statistics seedling rate.The calculation formula of seedling rate is: seedling rate=(seedling strain number)/(number seeds of inoculation).Experimental group: 90%.Control group: 75%.
2), detect the fungal infection situation of sprouting seedling.Experimental technique: begin during from access bacterium piece after cultivation is carried out 45-60 days altogether, to detect fungi and whether infect successfully the meter.The root segment of the seedling of experimental group and control group is put into respectively the 10%KOH aqueous solution, 121 ℃ of decolouring 20min, after taking out with aseptic water washing 3 times, with the 4h that dyes under 90 ℃ of conditions of aniline blue, alkaline H
2O
2Decolouring 8min catches the mycorhiza that is the fungal infection success of color in the root segment after the dehydration.The result: the experimental group root segment is caught color, and the control group root segment is not caught color, and the fungal infection success is described.3 repetitions are all established in experiment, and the result is all consistent.
3), estimate seedling resistance.Experimental technique: begin during from access bacterium piece the meter, after cultivation is carried out 45-60 days altogether, cultivate tissue culture plant inoculation after 45-60 days in the symbiotic culture medium that contains the different Cu ionic concn (oat medium) with common, cultivate 90 days (when the substratum with copper ions begins to cultivate, counting), observe the upgrowth situation of seedling.Be total to culture condition: intensity of illumination is 2500Lx, and the photoperiod is that 14h illumination/10h is dark, 25 ℃ of temperature.With the tissue cultured seedling of Mycorrhizal not in contrast.
Copper ion concentration is respectively 0.0064mg/L, 0.0128mg/L, 0.0192mg/L, 0.0256mg/L in the symbiotic culture medium.
Experimental result: Herba Dendrobii with bacterial strain DX08 symbiosis culture after for some time, when copper ion concentration was between 0.0192mg/L-0.0256mg/L in the substratum, plant strain growth was good, robust plant shows resistance; Control group plant spire jaundice, along with the variation of time from the yellow of spire top to whole plant, final dead, non-resistant.3 repetitions are established in experiment, and the result is all consistent.
Embodiment 3, cultivation Herba Dendrobii
One, the collection of Seeds of Dendrobium Candidum and pre-treatment: with consistent described in the embodiment 2.
Two, the preparation of bacterium piece: except incubation time in the DE substratum is 10 beyond the highest heavens, all the other steps are all with consistent described in the embodiment 2.
Three, cultivate altogether
Seeds of Dendrobium Candidum after 100 sterilizations is seeded in 5cm
2In the oat medium, place the long-pending 2mm of being of 1 block to media surface after planting simultaneously
3The bacterium piece of ball chamber bacterium (Mycosphaerella sp.) DX08CGMCC No.4149; Be that 22 ℃, periodicity of illumination are that 14h illumination/10h dark and intensity of illumination are to cultivate for 5 weeks under the condition of 2000Lx in temperature, obtain the Herba Dendrobii seedling.
The composition of oat medium (OMA): with identical described in the embodiment 2.
Four, result
The statistics seedling rate.With experimental group result among the embodiment 2 without significant difference.
Detect the fungal infection situation of sprouting seedling.The result: with experimental group result among the embodiment 2 without significant difference.
Estimate seedling resistance.The result: with experimental group result among the embodiment 2 without significant difference.
Embodiment 4, cultivation Herba Dendrobii
One, the collection of Seeds of Dendrobium Candidum and pre-treatment: with consistent described in the embodiment 2.
Two, the preculture of bacterial classification: except incubation time in the DE substratum is 20 beyond the highest heavens, all the other steps are all with consistent described in the embodiment 2.
Three, cultivate altogether
Seeds of Dendrobium Candidum after 200 sterilizations is seeded in 5cm
2In the oat medium, place the long-pending 2mm of being of 2 blocks to media surface after planting simultaneously
3The bacterium piece of ball chamber bacterium (Mycosphaerella sp.) DX08CGMCC No.4149; Be that 24 ℃, periodicity of illumination are that 14h illumination/10h dark and intensity of illumination are to cultivate for 5 weeks under the condition of 3000Lx in temperature, obtain the Herba Dendrobii seedling.
The composition of oat medium (OMA): with identical described in the embodiment 2.
Four, result
The statistics seedling rate.With experimental group result among the embodiment 2 without significant difference.
Detect the fungal infection situation of sprouting seedling.The result: with experimental group result among the embodiment 2 without significant difference.
Estimate seedling resistance.The result: with experimental group result among the embodiment 2 without significant difference.
Claims (11)
1. ball chamber bacterium (Mycosphaerella sp.) DX08, its deposit number is CGMCC N0.4149.
2. the application of ball chamber bacterium (Mycosphaerella sp.) DX08 CGMCC No.4149 in cultivating orchid; Described orchid is Dendrobium Sw; Described Dendrobium Sw is Herba Dendrobii (Dendrobium officinale).
3. a method of cultivating orchid comprises the steps: Orchid Seeds and ball chamber bacterium (Mycosphaerella sp.) DX08 CGMCC No.4149 are cultivated altogether, obtains the orchid seedling; Described orchid is Dendrobium Sw; Described Dendrobium Sw is Herba Dendrobii (Dendrobium officinale).
4. method according to claim 3, it is characterized in that: described method of cultivating altogether comprises the steps: described Orchid Seeds is seeded in the substratum, place again the cultivation bacterium piece of ball chamber bacterium (Mycosphaerella sp.) DX08 CGMCC No.4149 to media surface after planting, cultivate again.
5. method according to claim 4, it is characterized in that: the amount of described sowing is every 5cm
2Substratum in the sowing 100-200 described Orchid Seeds; The amount of the cultivation bacterium piece of described placement ball chamber bacterium (Mycosphaerella sp.) DX08CGMCC No.4149 is every 5cm
2Media surface place that the 1-2 block is long-pending to be 2mm
3Described cultivation bacterium piece.
6. method according to claim 5, it is characterized in that: the amount of described sowing is every 5cm
2Substratum in the sowing 100 or 150 or 200 described Orchid Seeds.
7. arbitrary described method according to claim 4-6 is characterized in that: described substratum is oat medium;
1 liter of described oat medium is comprised of 2.5g oatmeal, 8g agar and water, and water is supplied volume; The pH value of described oat medium is 5.8;
The cultivation bacterium piece of described ball chamber bacterium (Mycosphaerella sp.) DX08CGMCC No.4149 is to obtain according to the method that comprises the steps: ball chamber bacterium (Mycosphaerella sp.) DX08 CGMCC No.4149 is inoculated in the fungi culture medium, 25 ℃ of lower dark cultivations 10 days-20 days, get the bacterium piece from the bacterium colony center.
8. method according to claim 7, it is characterized in that: the cultivation bacterium piece of described ball chamber bacterium (Mycosphaerella sp.) DX08 CGMCC No.4149 is to obtain according to the method that comprises the steps: ball chamber bacterium (Mycosphaerella sp.) DX08 CGMCC No.4149 is inoculated in the fungi culture medium, 25 ℃ of lower dark cultivations 10 days or 15 days or 20 days, get the bacterium piece from the bacterium colony center.
9. arbitrary described method according to claim 3-6 is characterized in that: described temperature of cultivating altogether is 22 ℃-24 ℃, and the described periodicity of illumination of cultivating altogether is that 14h illumination/10h is dark, and described intensity of illumination of cultivating altogether is 2000Lx～3000Lx.
10. method according to claim 9 is characterized in that: described temperature of cultivating altogether is 22 ℃ or 23 ℃ or 24 ℃, and described intensity of illumination of cultivating altogether is 2000Lx or 2500Lx or 3000Lx.
11. method according to claim 7 is characterized in that: described fungi culture medium is the DE substratum; And/or the described time of cultivating altogether was 5 weeks.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101124888A (en) * | 2007-09-28 | 2008-02-20 | 南开大学 | Method for cultivating tetraploid of dendrobium officinale |
| CN101338290A (en) * | 2008-08-12 | 2009-01-07 | 金辉 | Method for culturing orchid special strain thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101124888A (en) * | 2007-09-28 | 2008-02-20 | 南开大学 | Method for cultivating tetraploid of dendrobium officinale |
| CN101338290A (en) * | 2008-08-12 | 2009-01-07 | 金辉 | Method for culturing orchid special strain thereof |
Non-Patent Citations (1)
| Title |
|---|
| 章玉平.兰花种子繁殖技术概况.《中国花卉园艺》.2003,(第04期), * |
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