CN106386483B - A kind of rapid propagation method for the leaf blueness moss that wrinkles - Google Patents
A kind of rapid propagation method for the leaf blueness moss that wrinkles Download PDFInfo
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 10
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- 230000002062 proliferating effect Effects 0.000 claims abstract description 5
- 239000000463 material Substances 0.000 claims description 18
- 239000002028 Biomass Substances 0.000 claims description 15
- 238000004659 sterilization and disinfection Methods 0.000 claims description 11
- 239000001963 growth medium Substances 0.000 claims description 10
- 238000011081 inoculation Methods 0.000 claims description 6
- 229920001817 Agar Polymers 0.000 claims description 5
- 239000008272 agar Substances 0.000 claims description 5
- 239000007836 KH2PO4 Substances 0.000 claims description 4
- NGPGDYLVALNKEG-UHFFFAOYSA-N azanium;azane;2,3,4-trihydroxy-4-oxobutanoate Chemical compound [NH4+].[NH4+].[O-]C(=O)C(O)C(O)C([O-])=O NGPGDYLVALNKEG-UHFFFAOYSA-N 0.000 claims description 4
- SURQXAFEQWPFPV-UHFFFAOYSA-L iron(2+) sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Fe+2].[O-]S([O-])(=O)=O SURQXAFEQWPFPV-UHFFFAOYSA-L 0.000 claims description 4
- 239000002609 medium Substances 0.000 claims description 4
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 4
- 238000007747 plating Methods 0.000 claims description 4
- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Inorganic materials [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 claims description 4
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- 241000208340 Araliaceae Species 0.000 claims 1
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- 235000008434 ginseng Nutrition 0.000 claims 1
- 238000004519 manufacturing process Methods 0.000 abstract description 3
- 241000196324 Embryophyta Species 0.000 description 7
- 238000011160 research Methods 0.000 description 4
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 3
- 238000009395 breeding Methods 0.000 description 3
- 230000001488 breeding effect Effects 0.000 description 3
- 229910052802 copper Inorganic materials 0.000 description 3
- 239000010949 copper Substances 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 3
- 229910052737 gold Inorganic materials 0.000 description 3
- 239000010931 gold Substances 0.000 description 3
- 230000001954 sterilising effect Effects 0.000 description 3
- 241000514966 Brachythecium kuroishicum Species 0.000 description 2
- 238000000692 Student's t-test Methods 0.000 description 2
- 206010000210 abortion Diseases 0.000 description 2
- 231100000176 abortion Toxicity 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
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- 229930002875 chlorophyll Natural products 0.000 description 2
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- QPLDLSVMHZLSFG-UHFFFAOYSA-N Copper oxide Chemical compound [Cu]=O QPLDLSVMHZLSFG-UHFFFAOYSA-N 0.000 description 1
- 239000005751 Copper oxide Substances 0.000 description 1
- 229910004616 Na2MoO4.2H2 O Inorganic materials 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- 239000005708 Sodium hypochlorite Substances 0.000 description 1
- 241000592342 Tracheophyta Species 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 230000003044 adaptive effect Effects 0.000 description 1
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- 238000013401 experimental design Methods 0.000 description 1
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- 239000012499 inoculation medium Substances 0.000 description 1
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- 238000005259 measurement Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 238000005065 mining Methods 0.000 description 1
- 239000002574 poison Substances 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 231100000241 scar Toxicity 0.000 description 1
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 description 1
- FDEIWTXVNPKYDL-UHFFFAOYSA-N sodium molybdate dihydrate Chemical compound O.O.[Na+].[Na+].[O-][Mo]([O-])(=O)=O FDEIWTXVNPKYDL-UHFFFAOYSA-N 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
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- 239000004575 stone Substances 0.000 description 1
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- RZLVQBNCHSJZPX-UHFFFAOYSA-L zinc sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Zn+2].[O-]S([O-])(=O)=O RZLVQBNCHSJZPX-UHFFFAOYSA-L 0.000 description 1
Classifications
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- 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
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- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Developmental Biology & Embryology (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- Environmental Sciences (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention discloses a kind of rapid propagation methods for the leaf blueness moss that wrinkles.Include the following steps：Field acquisition wrinkle leaf blueness moss cauline leaf gametophyte and sporangium, sterile water impregnate 5 hours；Gametophyte is cut into 1 centimetre of segment after rinsing well, the hypochlorite disinfectant 1 minute in the sterile small culture dish of 5 centimetres of diameter, polishing, which crushes, after sterile water wash 5 times is inoculated in BCD culture mediums, while sporangium is placed in 1.5ml centrifuge tubes and sterilizes 5 minutes, and BCD culture mediums are inoculated in after being pulverized with tweezers；It is 23 DEG C in temperature, 80 μm of ol photons m‑2s‑1Light intensity is grown 2 weeks in the incubator under 16 hour long-day；After gametophyte and spore grow new protonema, BCD culture mediums are inoculated in after mechanical grinding crushes, prolific protonema is can obtain within one week, obtains ripe gametophyte within one month.Engineering production for later stage wrinkle leaf blueness moss provides technical guarantee.
Description
Fields
The invention belongs to biotechnologies, and in particular to a kind of wrinkle leaf blueness moss protonema and the sterile quick breeding of gametophyte
Method.
Background technology
Moss is a kind of from the aquatic higher plant monoid to terrestrial transition, and the whole world spreads all over there are about 21200 kinds except ocean
It every nook and cranny or even is grown on desert, tundra and rock on the outer earth, drought-enduring, cold-resistant, impoverishment tolerant, adaptive faculty is extremely strong, is
The pioneer of the Nature and pioneer (Kidron, 2014；Cao is equal, and 2014).The moss resource in Yunnan is especially abundant, has full generation
The moss kind of boundary treaty 36%.Large-scale Copper and the investigation of plant resources in the serious area of gold mine ecological environment destruction are saved to China more
It was found that the grown that moss can not survive as pionner in these vascular plants.For example, in Dongchuan District, Yunnan Province tangdan copper mine
Green moss, 6 sections of Yu Xiandeng, 22 kinds of bryophytes are dispersed with, wherein on the ore-rock stone for directly live in patination, in addition 68% is
32% is lived on the ore deposit soil containing copper oxide.Yunnan wall moss etc. is widely distributed in the southwest of Guizhou Province Lateritic Gold Deposit, Dongchuan District, Yunnan Province drags
Bu Ka-broadcast card gold mine and tangdan copper mine area (Zhou＆Zhang, 2007；Jiang Hong and Zhang Chaohui, 2012), the especially center in mining area
Area destroys the place of most serious, and the growth of other plant is can't see other than fragmentary moss.
Wrinkle leaf blueness moss (BrachytheciumkuroishicumBesch.) is the widely distributed one kind of Yunnan hillside, scar
Moss, habitat is extremely wide, and the moist dark and sun-drenched place in meadow of hayashishita can be grown.Branches and leaves are long and narrow, can grow to 20 lis
Rice, has the advantages that impoverishment tolerant, high temperature resistant, low temperature etc. are a variety of, the growth and breeding period is very short, only 1 half a month, with extreme drought resisting
The breeding cycle of nearly 1 year such as moss kind gable moss or the red moss of tooth rib, which is compared, has the production advantage.The application of moss is led at present
A large amount of excavations in field are stayed in, on the one hand cause the destruction of natural environment and the scarcity of wild resource；On the other hand it causes
Kind is obscured, and the later stage breeds and research is difficult because the differences of Physiological of different cultivars causes.Sporangium is only handed in temperature
It is generated, but abortion rate is higher during for variation.It realizes that large-scale artificial propagation is relatively difficult, also has no any the relevant technologies at present
The report of method.If wrinkle leaf blueness moss commercially produced, tissue culture technique will push its scale efficiently to breed
One of important technology.
Invention content
The present invention is intended to provide a kind of wrinkle leaf blueness moss aseptic and rapid propagation method, the disinfection including gametophyte and sporangium,
Culture, protonema subculture and expansion.Purpose be for wrinkle leaf blueness moss engineering production or the adverse circumstance adaptability that logs in of research moss into
Change mechanism provides technical foundation.In view of the natural growth of wrinkle leaf blueness moss, more than summer and autumn, sunshine, light intensity and temperature reference are natural
Condition sets following artificial culture parameters.
In order to realize the above-mentioned purpose of the present invention, the present invention provides following technical solutions：
A kind of rapid propagation method for the leaf blueness moss that wrinkles, this method include the following steps：After gametophyte and sporangium disinfection, connect
Kind is cultivated in BCD culture mediums under certain condition of culture, then carries out subculture and the expansion of protonema, record growth feelings
Condition and biomass.
A kind of rapid propagation method for the leaf blueness moss that wrinkles as mentioned, wherein the gametophyte and sporangium disinfection are fields
Acquisition wrinkle leaf blueness moss cauline leaf gametophyte and sporangium, sterile water impregnate 5 hours, and gametophyte is cut into 1 centimetre of segment after rinsing well,
1.5% hypochlorite disinfectant 1 minute in the sterile small culture dish of 5 centimetres of diameter, after sterile water wash 5 times polishing crush inoculation
In BCD culture mediums, while sporangium is placed in 1.5ml centrifuge tubes and sterilizes 5 minutes, and BCD culture mediums are inoculated in after being pulverized with tweezers.
A kind of rapid propagation method for the leaf blueness moss that wrinkles as mentioned, wherein the inoculation is to carry out wrinkle leaf using Syrup-homogenizing instrument
The mechanical grinding of green moss, transfer 2ml is inoculated in 6 ware culture mediums after 1g vegetable material mixing 12ml sterile waters crush, and is placed in 16h long
Sunshine incubator, Syrup-homogenizing instrument use 10s/ times, and it is numerous soon with subculture that 3 times/material polishing parameter carries out material polishing.
A kind of rapid propagation method for the leaf blueness moss that wrinkles, wherein culture medium setting are used as mentioned：Utilize BCD basal mediums
As background culture medium, the BCD basal medium formulations are：MgSO4.7H21 μM of O, KH2PO418.4 μM, KNO310 μM,
FeSO4.7H2O 45μM；CuSO4.5H20.22 μM of O, H3BO310 μM, CoCl2.6H20.23 μM of O, Na2MoO4.2H2O 0.1μ
M, ZnSO4.7H20.19 μM of O, MnCl2.4H20.17 μM, ammonium tartrate 5mM of 2 μM of O, KI, agar 0.8%, 121 DEG C, 20min
Sterilizing.3%phytogel is added in plating medium.
A kind of rapid propagation method for the leaf blueness moss that wrinkles as mentioned, wherein the culture is to treat that gametophyte and sporangium disappear
After poison, gametophyte is inoculated in BCD culture mediums, and sporangium was inoculated in BCD culture mediums after being pulverized with tweezers and cultivates, by 16 hours
After long-day culture 2 weeks, gametophyte and spore grow new protonema, BCD culture mediums are uniformly inoculated in after mechanical grinding crushes
Tablet, culture obtain prolific protonema for one week, obtain ripe gametophyte within one month.
A kind of rapid propagation method for the leaf blueness moss that wrinkles as mentioned, wherein set during the culture three kinds of temperature and
The condition of culture of intensity gradient, three kinds of different temperatures gradients are：18 DEG C, 23 DEG C, 28 DEG C；Three kinds of different intensity gradients are：40μ
mol photons m-2s-1, 80 μm of ol photons m-2s-1, 120 μm of ol photons m-2s-1。
A kind of rapid propagation method for the leaf blueness moss that wrinkles as mentioned, wherein it is temperature 23 that condition of culture is set during culture
DEG C, 80 μm of ol photons m-2s-1Light intensity.
A kind of rapid propagation method for the leaf blueness moss that wrinkles as mentioned, wherein wrinkle leaf blueness moss protonema and gametophyte growth conditions
Observation carries out the protonema under different amplification and gametophyte growth using light microscope and the microscope with fluorescence channel
State observation, and photograph to record daily, compare branch and growth conditions.
A kind of rapid propagation method for the leaf blueness moss that wrinkles as mentioned, wherein the record growing state and biomass use
It collects under 9 kinds of condition of culture, each is no less than 5 ware materials, biomass is measured, with student ' s t-test statistical analyses.
The method of the present invention also can be described as：Field acquisition wrinkle leaf blueness moss cauline leaf gametophyte and sporangium, sterile water impregnate 5
Hour；Gametophyte is cut into 1 centimetre of segment after rinsing well, hypochlorite disinfectant 1 divides in the sterile small culture dish of 5 centimetres of diameter
Clock, polishing, which crushes, after sterile water wash 5 times is inoculated in BCD culture mediums, while sporangium is placed in 1.5ml centrifuge tubes and sterilizes 5 minutes,
BCD culture mediums are inoculated in after being pulverized with tweezers；After gametophyte and spore grow new protonema after 16 hour long-day cultivated 2 weeks,
BCD culture mediums are inoculated in after mechanical grinding crushes, this is second of subculture, and culture can obtain prolific precursor in one week
Body obtains ripe gametophyte in one month.Second of squamous subculture during the experiment set temperature and intensity gradient, are most preferably cultivated
Condition be 23 DEG C of temperature, 80 μm of ol photons m-2s-1Light intensity.Three kinds of different temperatures gradients are：18 DEG C, 23 DEG C, 28 DEG C.Three
Planting different intensity gradients is：40μmol photons m-2s-1, 80 μm of ol photons m-2s-1, 120 μm of ol photons m- 2s-1.BCD culture medium prescriptions used are:MgSO4.7H21 μM of O, KH2PO418.4 μM, KNO310 μM, FeSO4.7H2O 45μM；
CuSO4.5H20.22 μM of O, H3BO310 μM, CoCl2.6H20.23 μM of O, Na2MoO4.2H20.1 μM of O, ZnSO4.7H2O 0.19
μM, MnCl2.4H20.17 μM, ammonium tartrate 5mM of 2 μM of O, KI, agar 0.8%, 121 DEG C, 20min sterilizings.In plating medium
3%phytogel is added, instead of agar powder used in general culture medium, to facilitate microscopic observation.Wrinkle leaf blueness moss numerous material soon
Condition of culture is：23 DEG C, 16h illumination, 8h is dark, 80 μm of ol photons m of light intensity-2s-1Incubator.Wrinkle leaf blueness moss in field
Sodium hypochlorite used in disinfection a concentration of 1.5%.The multiple green and protonema of gametophyte is carried out by the microscopic observation of culture materials to grow
The judgement of state and record.The use of this method can with optimum condition promotion wrinkle leaf blueness moss protonema branch and growth,
The maximum amount of wrinkle leaf blueness moss protonema and gametophyte material are obtained in short time.
Description of the drawings
Fig. 1 is that present invention wrinkle leaf blueness moss gametophyte and sporangium disinfection are inoculated with microscope photo after growing 2 weeks；
Fig. 2 is the growth wrinkled under each temperature of leaf blueness moss and intensity gradient situation (bottom right angle staff=100 μm) after a week；
Fig. 3 is the biomass measuring to wrinkle under each temperature of leaf blueness moss and intensity gradient.
Specific embodiment
Below in conjunction with the accompanying drawings, the essentiality content further illustrated the present invention with the embodiment of the present invention, but not with
This limits the present invention.
Embodiment 1：
1. material and method：
1.1 research material：
The present invention is wrinkle leaf blueness moss (BrachytheciumkuroishicumBesch.) using material, during collecting location is
State's Kunming, Yunnan, specific location are Alt.1942m, 25 ° of 8 16 〞 of ˊ of north latitude, 102 ° of 44 38 〞 of ˊ of east longitude.
1.2 research method：
1.2.1 experimental design：Field acquisition wrinkle leaf blueness moss cauline leaf gametophyte and sporangium, sterile water impregnate 5 hours；Gamete
Body is cut into 1 centimetre of segment after rinsing well, 1.5% hypochlorite disinfectant 1 minute in the sterile small culture dish of 5 centimetres of diameter,
BCD culture mediums are inoculated in after sterile water wash 5 times, while sporangium is placed in 1.5ml centrifuge tubes and sterilizes 5 minutes, is pulverized with tweezers
After be inoculated in BCD culture mediums；After gametophyte and spore grow new protonema after 16 hour long-day cultivated 2 weeks, through mechanical grinding
BCD culture medium flat plates are uniformly inoculated in after crushing, culture can obtain prolific protonema in one week, obtain maturation within one month
Gametophyte.During the experiment set temperature and intensity gradient, three kinds of different temperatures gradients are：18 DEG C, 23 DEG C, 28 DEG C；Three kinds of differences
Intensity gradient is：40μmol photons m-2s-1, 80 μm of ol photons m-2s-1, 120 μm of ol photons m-2s-1.Most
Optimal culture condition is obtained eventually as 23 DEG C of temperature, 80 μm of ol photons m-2s-1Light intensity.It records under each condition of culture
Growing state and biomass.
1.2.2 protonema and gametophyte inoculation technique：The mechanical grinding of vegetable material, 1g plant materials are carried out using Syrup-homogenizing instrument
Transfer 2ml is inoculated in 6 ware culture mediums after material mixing 12ml sterile waters crush, and is placed in 16h long-day incubators, what the present invention used
Syrup-homogenizing instrument is：IKA (ULTRA TURRAX Tube Drive), using 10s/ times, 3 times/material polishing parameter carries out material polishing
It is numerous soon with subculture.
1.2.3 culture medium is set：By the use of BCD basal mediums as background culture medium, which is the present invention：
MgSO4.7H21 μM of O, KH2PO418.4 μM, KNO310 μM, FeSO4.7H2O 45μM；CuSO4.5H2O0.22 μM, H3BO3 10
μM, CoCl2.6H20.23 μM of O, Na2MoO4.2H20.1 μM of O, ZnSO4.7H20.19 μM of O, MnCl2.4H22 μM of O, KI 0.17
μM, ammonium tartrate 5mM, agar 0.8%, 121 DEG C, 20min sterilizings.3%phytogel is added in plating medium.
1.2.4 wrinkle leaf blueness moss protonema and the observation of gametophyte growth conditions：Using light microscope and with fluorescence channel
Microscope carries out protonema and the observation of gametophyte growth conditions under different amplification, and photographs to record daily, compares branch
And growth conditions.The microscope that the present invention uses is Kunming Inst. of Botany, Chinese Academy of Sciences's Biotechnology Platform fluorescence microscopy
Mirror, instrument model：Leica DM5500B.
1.2.5 wrinkle leaf blueness moss protonema biomass measuring statistics：Collect no less than 5 ware materials, analysis under 9 kinds of condition of culture
Balance measurement biomass, with student ' s t-test statistical analyses.
2. result and analysis
Form after 2.1 wrinkle leaf blueness moss gametophytes and sporangium disinfection inoculation are grown 2 weeks.
Wrinkle leaf blueness moss is haploid gametophyte plant dominant from generation to generation, and Somatic Embryogenesis is very competent, by plant
Inoculation medium can carry out amount reproduction under aseptic condition after the mechanical crushing of material.Sporangium only alternately becomes in temperature simultaneously
It is generated during change, but abortion rate is higher and generates the raw material of protonema.Newborn plant is cultivated after gametophyte or spore disinfection inoculation
Object is organized as the precursor volume morphing of wrinkle leaf blueness moss, and protonema is in media surface sprawl growth, passes through constantly cell branch
And cell elongation obtains the increase of biomass.Chlorophyll generates red fluorescence under ultraviolet excitation, is reacting cells vigor
Index.Explant after disinfection shows very faint red fluorescence because of chlorophyll degradation in fluorescence microscope, living
The vigorous cell of power has ripe chloroplaset, shows that (Fig. 1, white arrow indicate just inoculated explant to strong red fluorescence
Body position).
Growing state at each temperature of 2.2 wrinkle leaf blueness moss and intensity gradient
Set temperature and intensity gradient during second of polishing squamous subculture, the results show that 9 kinds of culture items after cultivating 1 week
Protonema amount reproduction is all detected under part, and is placed in 23 DEG C, 80 μm of ol photons m of light intensity-2s-1Incubator in when, wrinkle
The growth of leaf blueness moss protonema is most vigorous, 18 DEG C, 40 μm of ol photons m of light intensity-2s-1Condition of culture under grow most slow (figure
2).Illustrate that temperature and the intensity of illumination range of the moss adaptation of wrinkle leaf blueness are wider, but prefer higher temperature and luminous intensity.
Biomass measuring at each temperature of 2.3 wrinkle leaf blueness moss and intensity gradient
The biomass of growth in one week, as a result shows unanimously with Fig. 2 under 9 kinds of condition of culture of statistical analysis.23 DEG C, 80 μ of light intensity
mol photons m-2s-1Culture wrinkle leaf blueness moss protonema growth is most fast, 18 DEG C, 40 μm of ol photons m of light intensity-2s-1Training
It is grown under the conditions of supporting most slow.Abscissa shows light intensity, and ordinate shows biomass, and unit is milligram.Asterisk shows same light intensity
T-test detections are in p when the lower numerical value of degree is compared with 18 DEG C of biomass<Significant difference in 0.01 level；Data are expressed as putting down
Mean value ± standard error, n≤5 (Fig. 3).
Claims (4)
1. a kind of rapid propagation method for the leaf blueness moss that wrinkles, it is characterised in that this method includes the following steps：Gametophyte and sporangium
It after disinfection, is inoculated in BCD culture mediums, is cultivated under certain condition of culture, then carry out subculture and the expansion of protonema, into
Row wrinkle leaf blueness moss protonema and the observation of gametophyte growth conditions, and growing state and biomass are recorded, the gametophyte and spore
Ascus disinfection is field acquisition wrinkle leaf blueness moss cauline leaf gametophyte and sporangium, and sterile water impregnates 5 hours, after gametophyte is rinsed well
1 centimetre of segment is cut into, 1.5% hypochlorite disinfectant 1 minute, sterile water wash 5 times in the sterile small culture dish of 5 centimetres of diameter
Polishing, which crushes, afterwards is inoculated in BCD culture mediums, while sporangium is placed in 1.5ml centrifuge tubes and sterilizes 5 minutes, is inoculated with after being pulverized with tweezers
In BCD culture mediums,
The inoculation is that the mechanical grinding of wrinkle leaf blueness moss is carried out using Syrup-homogenizing instrument, and 1g vegetable material mixing 12ml sterile waters crush
Transfer 2ml is inoculated in 6 ware culture mediums afterwards, is placed in 16h long-day incubators, and Syrup-homogenizing instrument uses 10s/ times, 3 times/material polishing ginseng
It is numerous soon with subculture that number carries out material polishing；
The culture is after gametophyte and sporangium disinfection, and gametophyte is inoculated in BCD culture mediums, and sporangium is pulverized with tweezers
After be inoculated in BCD culture mediums and cultivate, cultivated 2 weeks by 16 hour long-day, after gametophyte and spore grow new protonema, pass through
Mechanical grinding is uniformly inoculated in BCD culture medium flat plates after crushing, culture obtains prolific protonema for one week, obtains within one month ripe
Gametophyte；
Culture medium setting is by the use of BCD basal mediums as background culture medium, and the BCD basal medium formulations are：
MgSO4.7H21 μM of O, KH2PO418.4 μM, KNO310 μM, FeSO4.7H2O 45μM；CuSO4.5H20.22 μM of O, H3BO3
10 μM, CoCl2.6H20.23 μM of O, Na2MoO4.2H20.1 μM of O, ZnSO4.7H20.19 μM of O, MnCl2.4H22 μM of O, KI
0.17 μM, ammonium tartrate 5mM, agar 0.8%, 121 DEG C, 20min sterilizes, and 3%phytogel is added in plating medium；
The condition of culture of three kinds of temperature and intensity gradient is set in the training period, and three kinds of different temperatures gradients are：18 DEG C, 23 DEG C,
28℃；Three kinds of different intensity gradients are：40μmol·m-2·s-1, 80 μm of olm-2·s-1, 120 μm of olm-2·s-1。
2. the rapid propagation method of a kind of leaf blueness moss that wrinkles as described in claim 1, it is characterised in that set during the culture
Condition of culture is put as 23 DEG C of temperature, 80 μm of olm-2·s-1Light intensity.
3. the rapid propagation method of a kind of leaf blueness moss that wrinkles as described in claim 1, it is characterised in that the wrinkle leaf blueness moss is former
Filament and the observation of gametophyte growth conditions are carried out using light microscope and the microscope with fluorescence channel under different amplification
Protonema and the observation of gametophyte growth conditions, and photograph to record daily, compare branch and growth conditions.
A kind of 4. rapid propagation method of leaf blueness moss that wrinkles as described in claim 1, it is characterised in that the record growth feelings
Using collecting under 9 kinds of condition of culture, each is no less than 5 ware materials, biomass is measured, with student ' s t- for condition and biomass
Test statistical analyses.
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| CN109937881A (en) * | 2019-04-21 | 2019-06-28 | 中国科学院昆明植物研究所 | A kind of rapid propagation method of bright leaf moss protonema and gametophyte |
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| CN102144545A (en) * | 2011-01-11 | 2011-08-10 | 上海师范大学 | In vitro rapid propagating method of brachythecium procumbens gametophyte |
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