CN102612966B - Method for obtaining dendrobium aphyllum seeds to germinate effective symbiotic fungi - Google Patents
Method for obtaining dendrobium aphyllum seeds to germinate effective symbiotic fungi Download PDFInfo
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- 241000233866 Fungi Species 0.000 title claims abstract description 67
- 244000131844 Dendrobium aphyllum Species 0.000 title claims abstract description 52
- 238000000034 method Methods 0.000 title claims abstract description 23
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- 230000031068 symbiosis, encompassing mutualism through parasitism Effects 0.000 claims description 30
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- 239000002609 medium Substances 0.000 claims description 10
- 238000007789 sealing Methods 0.000 claims description 10
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- 229920001778 nylon Polymers 0.000 claims description 9
- 235000013399 edible fruits Nutrition 0.000 claims description 8
- 239000004744 fabric Substances 0.000 claims description 8
- 230000001954 sterilising effect Effects 0.000 claims description 8
- 238000011081 inoculation Methods 0.000 claims description 7
- 239000008223 sterile water Substances 0.000 claims description 7
- 239000000725 suspension Substances 0.000 claims description 7
- 229920001817 Agar Polymers 0.000 claims description 6
- 239000008272 agar Substances 0.000 claims description 6
- 238000004659 sterilization and disinfection Methods 0.000 claims description 6
- 230000010152 pollination Effects 0.000 claims description 4
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- 244000061456 Solanum tuberosum Species 0.000 claims description 3
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- 230000004345 fruit ripening Effects 0.000 claims description 3
- 239000006481 glucose medium Substances 0.000 claims description 3
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- 210000004185 liver Anatomy 0.000 claims description 3
- 238000012544 monitoring process Methods 0.000 claims description 3
- 238000005406 washing Methods 0.000 claims description 3
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 claims description 2
- 241001632422 Radiola linoides Species 0.000 claims description 2
- 229910052801 chlorine Inorganic materials 0.000 claims description 2
- 239000000460 chlorine Substances 0.000 claims description 2
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- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 2
- 241000576531 Habenaria macroceratitis Species 0.000 description 2
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- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
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- 108091023242 Internal transcribed spacer Proteins 0.000 description 1
- 241000233805 Phoenix Species 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
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Abstract
The invention discloses a method for obtaining dendrobium aphyllum seeds to germinate effective symbiotic fungi. The method comprises the following steps of: subjecting parent dendrobium aphyllum plants to artificial cross-pollination to obtain mature seeds, putting the seeds into a special seed bag, placing the seed bag at an origin place where dendrobium aphyllum plants grow, and inducing the seeds under natural conditions to germinate and produce protocorms; cultivating the obtained protocorms in an artificial culture medium under sterile conditions after surfaces of the protocorms are sterilized, inducing the growth of endophytic fungi in the protocorms, and purifying the endophytic fungi to obtain pure bacterial colonies after hyphae grow out; and cultivating the obtained fungi and the dendrobium aphyllum seeds for symbiotic germination, setting a control experiment so as to test effectiveness of the obtained fungi to the germination of the dendrobium aphyllum seeds, screening the symbiotic fungi which are effective to germinate the dendrobium aphyllum seeds, subjecting the fungi to molecular identification, and storing the fungi. According to the method disclosed by the invention, a new way is found for the cultivation of seedlings through the symbiotic germination of the dendrobium aphyllum seeds and the fungi.
Description
Technical field
The present invention relates to obtain the method for the effective symbiosis fungi of germination of arethusa seeds, relate in particular to and a kind ofly induce seed germination at Dendrobium aphyllum (Roxb.) C. E. Fisch (Dendrobium aphyllum) Proterozoic, obtain protocorm, from protocorm, separate, cultivate and filter out the method to the effective symbiosis fungi of seed germination again.
Background technology
The seed of orchid is very tiny, and the embryo of ateliosis is only arranged, and seed germination need rely on specific symbiosis fungi to obtain nutriment under field conditions (factors).The sprouting of orchid seed at present, the synthetic mediums that adopt carry out non-symbiosis germination more under aseptic condition, though germination rate is high, but when carrying out the open-air recurrence of kind in imminent danger, sprigging survival rate in the natural environment is lower, thereby and causes subsequent growth seriously limited owing to can't set up symbiotic relation with the fungi of occurring in nature.Can solve this problem by seed and its effective fungi symbiotic germination under field conditions (factors) of sprouting.Effectively the acquisition of fungi many employings at present separate from the root of the wild plant of orchid.But owing to exist the endogenetic fungus of a large amount of effect the unknowns in the orchid root, this makes the screening of the effective fungi of seed germination become complicated and loaded down with trivial details with separating.Simultaneously, whether the fungi in the different orchid roots is identical still uncertain at present with effective symbiosis fungi in seed germination stage.Therefore, effective symbiosis fungi of acquisition seed germination is to carry out the key link that rare orchid in imminent danger returns.
Summary of the invention
The objective of the invention is at the deficiencies in the prior art, a kind of method that obtains the effective symbiosis fungi of Dendrobium aphyllum (Roxb.) C. E. Fisch seed germination is provided, create conditions for the field of realizing Dendrobium aphyllum (Roxb.) C. E. Fisch returns.
Purpose of the present invention is achieved by the following technical programs.
Except as otherwise noted, percentage of the present invention is percetage by weight.
Technical scheme of the present invention mainly is based on following understanding.
We put into special seed packet with the seed of Dendrobium aphyllum (Roxb.) C. E. Fisch, and seed packet is placed on the Proterozoic of Dendrobium aphyllum (Roxb.) C. E. Fisch plant strain growth, induce seed germination to produce protocorm, to under aseptic condition, use artificial medium culture behind the protocorm surface sterilizing that obtain, induce the endogenetic fungus growth in the protocorm, when waiting to grow mycelia, carry out the purifying of fungi, obtain pure bacterium colony, and carry out Molecular Identification and the preservation of fungi.The fungi of acquisition and the seed of Dendrobium aphyllum (Roxb.) C. E. Fisch are carried out the symbiotic germination cultivation, control experiment is set, with the validity of the fungi that obtained of check to the seed germination of Dendrobium aphyllum (Roxb.) C. E. Fisch, the result shows that the fungi that obtains is effective symbiosis fungi of Dendrobium aphyllum (Roxb.) C. E. Fisch seed germination, thereby has realized purpose of the present invention.
A kind of method that obtains the effective symbiosis fungi of Dendrobium aphyllum (Roxb.) C. E. Fisch seed germination may further comprise the steps:
(1) carry out artificial different friendship pollination at the Dendrobium aphyllum (Roxb.) C. E. Fisch florescence, results fruit when fruit maturation but fruit pod are not split as yet is placed on seed drying in the aseptic airtight glass container and preserves under-20 ℃ of conditions, and is stand-by;
(2) tweezers of usefulness sterilization on nylon net cloth, with having broadcast the nylon net cloth doubling of seed, get up with heat sealing machine the planting seed of 100～200 Dendrobium aphyllum (Roxb.) C. E. Fischs with three bandings, and the seed packet that completes is sewed on label at the seed packet edge, are convenient to monitoring and recovery;
(3) selection has the different location of the adult plant self-sow of Dendrobium aphyllum (Roxb.) C. E. Fisch, and the seed packet fixed placement near the trunk the plant root of growing up near Dendrobium aphyllum (Roxb.) C. E. Fisch, is covered one deck liver moss and preserves moisture on seed packet;
Should try one's best near the plant root of growing up when seed packet is placed, and seed packet is the level of state, it is agglomerating to avoid seed to assemble under the gravity effect.
(4) regularly reclaim 2～3 seed packets from each set-point every month, check the sprouting situation of seed in the seed packet, when finding protocorm is arranged, reclaim all seed packets;
(5) protocorm that reclaims is washed with running water successively, liquor natrii hypochloritis's sterilization cuts in half after the rinsed with sterile water, and cut sides is affixed on the potato glucose medium PDA and cultivates;
When (6) the protocorm incision grows mycelia, with the aseptic inoculation pin constantly the mycelia of picking fungus colony edge to new PDA medium, shift 3～5 times after, obtain pure bacterium colony;
(7) seed of Dendrobium aphyllum (Roxb.) C. E. Fisch is put into sterile water and make uniform suspension, two aseptic filter papers are placed on culture dish surface at the oat agar medium (OMA) for preparing, and the seed suspension of drawing 150 microlitres respectively is dispersed on two filter paper bars equably; Contain the agar block of the single symbiosis fungi pure culture of separate sources in medium center inoculation, the control treatment group that does not connect bacterium is set simultaneously, each handles 10 repetitions, seals to be placed in the climatic cabinate and cultivates with sealing film;
(8) observe the seed of 1 different disposal weekly, record seed germination quantity and sprout time, seed germination and protocorm developmental state are carried out classification, record the quantity of interior seed of each stage or protocorm, by with the contrast that does not connect the bacterium processed group, filter out effective symbiosis fungi of Dendrobium aphyllum (Roxb.) C. E. Fisch seed germination.
In the above-mentioned steps, it is the nylon net cloth of 45 μ m that described seed packet adopts mesh.Such mesh size both can keep seed not drop out, and the mycelia of water and fungi is passed freely through, and should avoid damaging seed when heat sealing machine seals.
The seedling place that seed packet set-point described in the step (3) is grown up plant and upgraded naturally for Dendrobium aphyllum (Roxb.) C. E. Fisch is arranged simultaneously.
Seed packet after step (4) reclaims rinses out attachment and earth gently with running water, seed packet after cleaning is placed in the middle of the two-layer face tissue, applying light makes face tissue blot the seed packet redundant moisture, seal with careful the cutting off of scissors, open seed packet and be placed on observation seed germination situation under the anatomical lens.
The described liquor natrii hypochloritis's available chlorine of step (5) ion concentration is 1%, sterilizes aseptic water washing 3～4 times 3～5 minutes; This step arranges control treatment simultaneously, do not cut behind the protocorm surface sterilizing with same source and directly place the PDA medium, if do not grow fungi in the control group medium, illustrate that the fungi that grows from experimental group protocorm otch is the interior living symbiosis fungi of protocorm on every side.Culture dish is put into climatic cabinate with sealing after film is sealed, 25 ± 2 ℃ of dark culturing.
The suspension of described 150 microlitres of step (7) contains 150 Dendrobium aphyllum (Roxb.) C. E. Fisch seeds approximately, and described condition of culture is: intensity of illumination is 2000Lx-3000Lx, 25 ℃ ± 2 ℃ of temperature, photoperiod 12h/12hL/D.
The described seed germination of step (8) and protocorm developmental state are with reference to Stewart ﹠amp; The method of Zettler (2002) is carried out classification to seed germination and protocorm developmental state, and be divided into 5 stages: it is transparent that the stage 0 is described as embryo, and the kind skin is intact, and seed is not sprouted, and the stage 1 is described as the embryo imbibition; Stage 2 is described as embryo continues to expand, and prominent breaking in the seed coat is considered as sprouting; Stage 3 is described as occurring protomeristem; Stage 4 is described as growing first blade; Stage 5 is described as first blade continued growth, and is elongated.
Stewart SL, Zettler LW (2002) Symbiotic germination of three semi-aquatic rein orchids (Habenaria repens, H.quinqueseta, H.macroceratitis) from Florida. (the beautiful phoenix flower plant of the three kinds of semi-aquatics in Florida (Habenaria repens, H.quinqueseta, H.macroceratitis) Aquatic Botany (aquatic botany) seed symbiotic germination), 72,25-35.
The effective symbiosis fungi of the Dendrobium aphyllum (Roxb.) C. E. Fisch seed germination that obtains is carried out Molecular Identification and comparison, it is glued membrane Pseudomonas (Tulasenlla) fungi, and with the database (NCBI of the U.S. state-run biotechnology information centre, http://www.ncbi.nlm.nih.gov/) accession number is that the fungi Tulasenlla calospora of GU166410.1 is the most similar in, maximum similarity reaches 99%, it is numbered FDaI7, adopts the test tube slant method to be stored in Xishuangbanna Tropical Plant Garden, Chinese Academy of Sciences.In the described Molecular Identification, adopt the CTAB method to extract fungal DNA, the pcr amplification the primer is ITS1 and ITS4; Pcr amplification product is that Shanghai living worker's biotechnology Co., Ltd checks order.
The present invention has the following advantages with respect to prior art: though the separating method of the effective symbiosis fungi of the relevant germination of arethusa seeds with bibliographical information of patent is arranged both at home and abroad, adopt of these methods separated fungi more from the root of the wild plant of orchid.Owing to exist the endogenetic fungus of a large amount of effect the unknowns in the orchid root, this makes the screening of the effective symbiosis fungi of seed germination and mask work unusual complicated and loaded down with trivial details.Simultaneously, whether the fungi in the different orchid roots is identical and uncertain with effective symbiosis fungi in seed germination stage, and it is very difficult to obtain the effective symbiosis fungi of seed germination.Separation to the effective symbiosis fungi of Dendrobium aphyllum (Roxb.) C. E. Fisch seed germination does not still have report.Method of the present invention is directly separated fungi from the protocorm of Dendrobium aphyllum (Roxb.) C. E. Fisch seed original place symbiotic germination, simple to operate, the fungal species that obtains is single, do not need a large amount of loaded down with trivial details screening processes, a kind of glued membrane Pseudomonas fungi that is separated to is by testing with the symbiotic germination of seed, prove effective symbiosis fungi of Dendrobium aphyllum (Roxb.) C. E. Fisch seed germination, thereby open up a new way for utilizing Dendrobium aphyllum (Roxb.) C. E. Fisch seed and mycosymbiosis to sprout to cultivate seedling.
Embodiment
By the following examples the present invention is described in further detail, but embodiment is not the restriction to technical solution of the present invention.All all should belong to protection scope of the present invention based on the conversion that training centre of the present invention is done.
Embodiment 1:
Choose healthy and strong plant when Dendrobium aphyllum (Roxb.) C. E. Fisch is bloomed and carry out artificial different friendship pollination, the about 180 days fruit maturations in pollination back, the fruit ripe but that the fruit pod is not split as yet of gathering, the surface is with 75% alcohol disinfecting and with cutting fruit with sterile razor blade after the sterile water wash, place the drier inner drying that fills silica gel after 24 hours in seed, in aseptic airtight vial, be stored in-20 ℃.
Tweezers with sterilization are evenly sowed the seed of 100-200 grain Dendrobium aphyllum (Roxb.) C. E. Fisch at 6cm * 9cm, mesh is on the nylon net cloth of 45 μ m, the nylon net cloth doubling of seed will have been broadcast, with heat sealing machine three bandings are got up, a seed packet completes, sew on plastic label at the seed packet edge, be convenient to monitoring and recovery.
Choose 3 seed packet set-points of 4 seed packet set-points and the green stone forest of Xishuangbanna Nature Reserve forest parks in Xishuangbanna Tropical Plant Garden, Chinese Academy of Sciences, amount to 7 seed packet set-points.These set-points of choosing have the adult plant of Dendrobium aphyllum (Roxb.) C. E. Fisch self-sow, and plant growth condition is good, can normally blossom and bear fruit.Each set-point is respectively placed 30 seed packets, amounts to 210 seed packets, and seed packet is numbered.Seed packet is placed near on near the trunk the adult plant root of Dendrobium aphyllum (Roxb.) C. E. Fisch, fixes with nylon wire, on seed packet, cover one deck liver moss, to play the effect of preserving moisture.Seed packet near the plant root of growing up, is the level of state seed packet as far as possible during placement, and it is agglomerating to avoid seed to assemble under the gravity effect.
Seed packet is placed after two months, respectively reclaim 2-3 seed packet from different set-points every month, seed packet after the recovery rinses out attachment and earth gently with running water, seed packet after cleaning is placed in the middle of the two-layer face tissue, applying light makes face tissue blot the seed packet redundant moisture, seal with careful the cutting off of scissors, open seed packet and be placed on observation seed germination situation under the anatomical lens.When finding protocorm is arranged, reclaim all remaining seed packets on August 20th, 2011.Obtain 55 protocorms altogether from 4 set-points, do not have protocorm or seed packet breakage in all the other 3 set-point seed packets.
Protocorm with after the running water flushing, is put in superclean bench and is filled the beaker that the available chlorine ion concentration is 1% liquor natrii hypochloritis, shake beaker 3-5 minute gently after, with aseptic blunt nosed tweezers taking-up, use aseptic water washing 3-4 time again.With sterile razor blade each protocorm is cut in half, cut sides is affixed on the ready potato glucose medium (PDA) and cultivates.Simultaneously the protocorm of 2 surface sterilizations is not cut and directly place the PDA medium, in contrast.Culture dish is put into climatic cabinate with sealing after film is sealed, 25 ± 2 ℃ of dark culturing.Cultivate after 7 days, do not grow mycelia around the protocorm of Qie Kaiing, and grow white hypha around the protocorm that cuts, can be defined as the endogenetic fungus of Dendrobium aphyllum (Roxb.) C. E. Fisch protocorm thus.
When the protocorm incision grows mycelia, with the aseptic inoculation pin constantly the mycelia of picking fungus colony edge to new PDA medium, shift 3-5 time after, get pure bacterium colony.
The Dendrobium aphyllum (Roxb.) C. E. Fisch seed of preserving is taken out from-20 ℃, and placing spends the night under the room temperature makes seed reply room temperature.Be that 1% liquor natrii hypochloritis sterilizes to seed and with after rinsed with sterile water 3-4 time, seed put into sterile water make uniform suspension with the available chlorine ion concentration.Place two 1.5cm * 4cm size aseptic filter paper on the culture dish surface of the oat agar medium (OMA) for preparing, draw 150 microlitre seed suspension (about 150) respectively and be dispersed in uniformly on two filter paper bars.At the about 0.5cm of medium center inoculation
3(1cm * 1cm * 0.5cm) contains the agar block of the single symbiosis fungi pure culture of separate sources, the control treatment group that does not connect bacterium is set simultaneously, each handles 10 repetitions, be placed on and cultivate condition of culture in the climatic cabinate and be with sealing film sealing: intensity of illumination is 2000Lx-3000Lx, 25 ℃ ± 2 ℃ of temperature, photoperiod 12h/12h L/D.
After cultivating for 5 weeks, the seed germination that has only inoculation FDaI7 bacterial strain to handle reaches stage 2 and follow-up phase, the seed ratio that reaches stage 2 and follow-up phase is 48.79%, have only imbibition to reach the seed in stage 1 and inoculate FCb4 bacterial strain, FDaI2 bacterial strain and do not connect the bacterium control group, do not form protocorm, illustrate that FDaI7 is effective symbiosis fungi of Dendrobium aphyllum (Roxb.) C. E. Fisch seed germination.
FDaI 7 bacterial strains that obtain are carried out Molecular Identification and comparison, it is glued membrane Pseudomonas (Tulasenlla) fungi, and with the database (NCBI of the U.S. state-run biotechnology information centre, http://www.ncbi.nlm.nih.gov/) accession number is that the fungi Tulasenlla calospora of GU166410.1 is the most similar in, and maximum similarity reaches 99%.Adopt the test tube slant method to be stored in Xishuangbanna Tropical Plant Garden, Chinese Academy of Sciences to this bacterial strain.
Claims (6)
1. method that obtains the effective symbiosis fungi of Dendrobium aphyllum (Roxb.) C. E. Fisch seed germination may further comprise the steps:
(1) carry out artificial different friendship pollination at the Dendrobium aphyllum (Roxb.) C. E. Fisch florescence, results fruit when fruit maturation but fruit pod are not split as yet is placed on seed drying in the aseptic airtight glass container and preserves under-20 ℃ of conditions, and is stand-by;
(2) tweezers of usefulness sterilization on nylon net cloth, with having broadcast the nylon net cloth doubling of seed, get up with heat sealing machine the planting seed of 100～200 Dendrobium aphyllum (Roxb.) C. E. Fischs with three bandings, and the seed packet that completes is sewed on label at the seed packet edge, are convenient to monitoring and recovery;
(3) selection has the different location of the adult plant self-sow of Dendrobium aphyllum (Roxb.) C. E. Fisch, and the seed packet fixed placement near the trunk the plant root of growing up near Dendrobium aphyllum (Roxb.) C. E. Fisch, is covered one deck liver moss and preserves moisture on seed packet;
(4) regularly reclaim 2～3 seed packets from each set-point every month, check the sprouting situation of seed in the seed packet, when finding protocorm is arranged, reclaim all seed packets;
(5) protocorm that reclaims is washed with running water successively, liquor natrii hypochloritis's sterilization cuts in half after the rinsed with sterile water, and cut sides is affixed on the potato glucose medium PDA and cultivates;
When (6) the protocorm incision grows mycelia, with the aseptic inoculation pin constantly the mycelia of picking fungus colony edge to new PDA medium, shift 3～5 times after, obtain pure bacterium colony;
(7) seed of Dendrobium aphyllum (Roxb.) C. E. Fisch is put into sterile water and make uniform suspension, place two aseptic filter papers on the culture dish surface of the oat agar medium for preparing, the seed suspension of drawing 150 microlitres respectively is dispersed on two filter paper bars equably; Contain the agar block of the single symbiosis fungi pure culture of separate sources in medium center inoculation, the control treatment group that does not connect bacterium is set simultaneously, each handles 10 repetitions, seals to be placed in the climatic cabinate and cultivates with sealing film;
(8) observe the seed of 1 different disposal weekly, record seed germination quantity and sprout time, seed germination and protocorm developmental state are carried out classification, record the quantity of interior seed of each stage or protocorm, by with the contrast that does not connect the bacterium processed group, filter out effective symbiosis fungi of Dendrobium aphyllum (Roxb.) C. E. Fisch seed germination.
2. the method for the effective symbiosis fungi of acquisition Dendrobium aphyllum (Roxb.) C. E. Fisch seed germination according to claim 1 is characterized in that: it is the nylon net cloth of 45 μ m that described seed packet adopts mesh.
3. the method for the effective symbiosis fungi of acquisition Dendrobium aphyllum (Roxb.) C. E. Fisch seed germination according to claim 1 is characterized in that: in the step (3), should try one's best near the plant root of growing up when seed packet is placed, and seed packet is the level of state.
4. the method for the effective symbiosis fungi of acquisition Dendrobium aphyllum (Roxb.) C. E. Fisch seed germination according to claim 1 is characterized in that: the seedling place that the seed packet set-point described in the step (3) is grown up plant and upgraded naturally for Dendrobium aphyllum (Roxb.) C. E. Fisch is arranged simultaneously.
5. the method for the effective symbiosis fungi of acquisition Dendrobium aphyllum (Roxb.) C. E. Fisch seed germination according to claim 1, it is characterized in that: the described liquor natrii hypochloritis's available chlorine of step (5) ion concentration is 1%, sterilizes aseptic water washing 3～4 times 3～5 minutes.
6. the method for the effective symbiosis fungi of acquisition Dendrobium aphyllum (Roxb.) C. E. Fisch seed germination according to claim 1, it is characterized in that: the described actual conditions that places climatic cabinate to cultivate of step (7) is: intensity of illumination 2000Lx-3000Lx, 25 ℃ ± 2 ℃ of temperature, photoperiod 12h/12h L/D.
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| CN103210842B (en) * | 2013-02-04 | 2014-08-27 | 四川深达生物科技有限公司 | Dendrobium candidum test-tube flowering and seed setting method |
| CN103238470B (en) * | 2013-04-27 | 2014-04-23 | 陕西师范大学 | Method for inducing endophytic fungi of orchid |
| CN103468588B (en) * | 2013-09-17 | 2015-05-27 | 中国科学院西双版纳热带植物园 | Tulasnella strain and application thereof in promotion of germination of Dendrobium aphyllum seeds |
| CN105613296A (en) * | 2016-01-25 | 2016-06-01 | 浙江欧银农业发展有限公司 | Tissue culture inoculating method for dendrobium officinale |
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