CN101486991B - Method for propagating Tortula muralis protonema by cell engineering cultivation - Google Patents
Method for propagating Tortula muralis protonema by cell engineering cultivation Download PDFInfo
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- CN101486991B CN101486991B CN2009100941470A CN200910094147A CN101486991B CN 101486991 B CN101486991 B CN 101486991B CN 2009100941470 A CN2009100941470 A CN 2009100941470A CN 200910094147 A CN200910094147 A CN 200910094147A CN 101486991 B CN101486991 B CN 101486991B
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Abstract
The invention relates to a method for cultivating and breeding pangenesis tortula protonema by using cell engineering, which belongs to the technical field of biology and comprises following concrete steps: a. mature spores are sterilized, and capsulea surface is sterilized for 0.5 to 1 minutes by a solution that contains 1.0% of mercury bichloride and 1.0% of a washing agent; b. germination is implemented on a solid culture medium, the spores are inoculated to an MS culture medium that has the pH value of 5.8 and is added with 2 to 5 mg/L of 2,4-D, 2.0g/L of sodium silicate and 2.0% of sucrose, and cultivated at the temperature of 25 plus or minus 3 DEG C and illumination intensity of 4000lx for 10 to 20 days; c. mass propagation of the protonema is implemented in the MS fluid culture medium for spore germination that has pH value of 5.8 and is added with 2 to 5 mg/L of 2,4-D, 2.0g/L of sodium silicate and 2.0% of sucrose, shaking cultivation is implemented at the temperature of 25 plus or minus 3 DEG C and the speed of 120rpm for 7 to 10 days and the power source is diffused lights; or augmentation cultivation is implemented in a fermentation flask charged with sterile air at the speed of 200L per hour; a small amount of the protonema is collected by 800rpm centrifugation, and a large amount of the protonema is collected through the filtration by 4 to 8 gauze layers. The invention has the advantages of simple and convenient preparation method, low cost and consistent maturity of the obtained protonema.
Description
Technical field:
The present invention relates to a kind of method of cultivating the general living wall moss protonema of breeding, belong to biological technical field with cell engineering.
Background technology:
In vegitabilia, bryophyte is the phyto-group that is transferred to Lu Sheng life transition type by the aquatic life of a class, is one of the most ancient population in the terrestrial plant on the earth.Comprise that except that above high mountain of 5000m and ocean, all there is the footprint of bryophyte in the South Pole in the different ecosystems on the earth at present.Bryophyte is under the jurisdiction of the spore plants in the higher plant, and the whole world has 23000 kinds approximately, and plant number and be only second to angiosperm, be an important class in the vegitabilia.
The life history of bryophyte, the common green bryophyte body of people belonged to gametophytic generation based on gametophyte.The sporophyte of bryophyte is grown nonparasitically upon another plant on gametophyte, sporophyte aplasia sometimes, and gametophyte reaches the differentiation of height usually.Have tangible digenesis the life history of bryophyte, experienced the three phases that early stage terrestrial plant is evolved its life history: the filament during immature gametophyte, grow up gametophytic cormus and diplontic sporophyte.Because bryophyte has simple relatively development models, the pattern of form differentiation is more simple, and (general n=6-10), they are suitable for cytodifferentiation analysis and morphologic research to hypoploidy, bryophyte life history is based on gametophyte, than being easier to carry out genetics research.
Bryophyte distributes very wide, move in the fresh water, the environment that can not break away from water, but also there are many kinds can restrain oneself drought environment extremely, be grown in above the exposed fully rock, it is only second to lichens to the suffertibility of the environment of arid, so bryophyte and lichens often are described as the pioneer of the Nature by the ecologist.
Wide owing to distribute, habitat diversification and to the idioadaptation mechanism of severe environment, most bryophytes can produce the secondary metabolites that enriches especially, and bryophyte is considered to the source of biologically active substance.Among the people, bryophyte is widely used in treating diseases such as wound, burn, infection, pulmonary tuberculosis, neurasthenia, convulsions, scald and pneumonia.In addition, nearly all bryophyte is not searched for food by various animal, do not infected by bacterium, fungi or virus yet, all these shows and exists some active substances in the liver moss as the autoprotection agent, resist extraneous destructive stimulus, for example, be sprayed at after the liver moss water homogenate that German farmer once collected with the limit, field and prevent insect pest on the Caulis et Folium Brassicae capitatae; Some composition of discovery liver mosses such as Lou Hongxiang can effectively be resisted HIV virus etc.Because bryophyte contains the broad variety compound, and chemical compound lot shows good biological activity, so bryophyte might become agricultural, the resources of medicinal plant that has a extensive future, and promotes the well-being of mankind.Yet the bryophyte body is little, mixes and grows thickly, be difficult for difference and separate between kind, the artificial culture and propagation of independent a kind of liver moss does not also have ripe system, and it is relatively more difficult again that some kind is gathered the sample of q.s, caused the research and development of bryophyte related products to face the raw material predicament.The tissue culture technique of plant exactly can solve this difficult problem, utilize quick breeding technology, can in the relatively short time, obtain the purified material of a large amount of single kind, promptly can be used for research, can be again and utilize liver moss to prepare various active ingredients a large amount of starting material are provided.
Reference:
[1]Stefan?A.Rensing，Daniel?Lang，Andreas?D.Zimmer，et?al.The?Physcomitrella?Genome?RevealsEvolutionary?Insights?into?the?Conquest?of?Land?by?Plants[J].Science，2008，319(5859)：64-69.
[2]D.Cove，M.Bezanilla，P.Harries，et?al.Mosses?as?model?systems?for?the?study?of?metabolism?anddevelopment[J].Annu?Rev?Plant?Biol，2006，57：497-520.
[3] Wu Pengcheng, bryophyte biology [M]. Beijing: Science Press, 1998.
[4] Wu Pengcheng, Jia Yu. geographical zone of Chinese bryophyte and distribution pattern [J]. plant resources and environment journal, 2006 (01): 1-8.
[5] Notherna barbarin tribes in ancient China is bright, muscology [M]. Beijing: Higher Education Publishing House, 1987.
[6]Becker?H.Zinsmeister?H.D.，Eicher?T.BryoPhytes，a?souree?of?biologically?active，naturally?occurringmaterial？[J].AngewChemintEdit，1991，30：130-147.
[7]Y.Asakawa，Recent?advances?in?phytochemistry?of?bryophytes-acetogenins，terpenoids?and?bis(bibenzyl)from?selected?Japanese，Taiwanese，New?Zealand，Argentinean?and?European?liverworts[M].New?York：Phytochemistry，2001.
[8]Asakawa?Y.Chemosystematics?of?the?Hepaticae[J].Phytochemistry，2004，65(6)：623-669.
[9] Wang Xiaoning, Lou Hongxiang. bryophyte bioactive ingredients progress [J]. herbal medicine, 2005 (02): 303-307.
[10] Wu Yuhuan, Cheng Guodong, high modest. ecological functions of bryophyte and effect [J] .20 in vegetation restoration and reconstruction, 2003,3:215-220.
[11] Wang Shidong, Bai Xueliang, Yong Shipeng. area, Shapotou bryoflora preliminary study [J]. Chinese eremophyte, 2001,21 (3): 244-249.
[12]Erikson?M，Miksche?G.E.On?the?occurrence?of?lignin?or?polyphenols?in?some?mosses?andliverworts.[J].Phytochemistry，1974，13：2295-2299.
[13] Bai Xueliang, Wang Yao, Xu Jie, breeding and the growth characteristics research [J] of area, et al. Shapotou dikaka crust layer moss. Chinese desert, 2003,23 (2): 171-176.
Summary of the invention:
The objective of the invention is to obtain by the method for cell engineering the general living wall moss protonema of a large amount of development degree unanimities, for developmental biology, extract efficacy study, ecological recovery provide a large amount of available materialss.
The present invention is achieved in that
One, the axenic germination of the ripe spore of general living wall moss obtains primary protonema
The acquisition of the aseptic primary protonema of the ripe spore of general living wall moss obtains via the sterilization of ripe spore, the sprouting on the solid medium, and concrete steps are as follows:
1. the sterilization of ripe spore:
Select the sporangium of general living wall moss mature and plump, from the following 1-2cm of sporangium, the seta middle portion is cut off, and cleans the sporangium surface with clear water, uses 75% alcohol-pickled sporangium then, sterilizes in advance tissue surface 0.5-1 minute; Then the solution with mercury chloride that contains 1.0g/L and 1.0% meal lotion carries out disinfection to the sporangium surface, and the treatment time is 3-5 minute; With aqua sterilisa soaking and washing sporangium 4 times repeatedly, the mercury chloride and the washing composition on cleaning material sporangium surface are residual again; With the fixing seta of aseptic nipper,, expose inner spore then, extrude spore in the centrifuge tube that fills sterilized water, make the spore dispersion suspension with tweezers with aseptic eye scissors transversely cutting sporangium top; The centrifuge tube good seal of spore will be filled, 40 ℃ water-bath moderate stimulation 40-50 second;
2. the sprouting on the solid medium:
Under aseptic condition, getting density is 10
4-10
6The spore of/ml, the MS substratum that is inoculated into improvement (has added in the 2,4 dichloro benzene ethoxyacetic acid (2,4-Dichlorophenoxyacetic acid is called for short 2,4-D) 2-5mg/L, water glass 2.0g/L, the MS substratum of sucrose 2.0%, pH5.8).Culture temperature is 25 ℃ ± 3 ℃, and illuminance is 4000lx, obtains the primary protonema by spore germination after 10-20 days.
Two, a large amount of breedings and the collection of protonema
1. a large amount of breedings and the collection of protonema:
A large amount of breedings of protonema and the operation of collecting by centrifugal purification behind the shake-flask culture obtain.Specific as follows:
(1) a large amount of breedings of protonema
With tweezers gently the gripping volume be 10-1000mm
3The protonema of sprouting, be inoculated into and added 2,4-D 2-5mg/L, water glass 2.0g/L, the volume of the MS liquid nutrient medium of sucrose 2.0%, pH5.8 is in the 50-1000ml triangular flask, and the nutrient solution that adds in each triangular flask is triangular flask volumetrical 1/3-1/2, and the 120rpm shaking table was cultivated 7-10 days, envrionment temperature is controlled at 25 ± 3 ℃, and light source is a diffused light; Carry out obtaining a large amount of protonemas after rolling bottle is cultivated 7-15 with identical substratum and control condition subsequently.The amount bigger as needs, then under aseptic condition, the protonema that cultivation obtains is poured in the glass fermentor tank together with nutrient solution, the fresh medium that adds 3-10 times of volume, the bottom feeds sterile air (200L/ hour), make the protonema in the nutrient solution constantly seethe, with the no permanent sedimentary protonema in fermentor tank bottom is standard, cultivates after 7-15 days, stops air inlet, treat protonema sink to jar at the bottom of after, the nutrient solution that slowly will be in tank body top with peristaltic pump under aseptic condition extracts 2/3 out, adds extremely originally 5 times of volume of fresh sterile nutrient solutions again, continues to repeat enlarged culturing, till the tank body maximum volume, promptly obtain more substantial protonema so repeatedly.
(2) collection of protonema
As required, the protonema of cultivating in the triangular flask can be used for the enlarged culturing that goes down to posterity, also the whole suspensions in the culturing bottle can be poured in the centrifuge tube, 800rpm/min (rev/min) centrifugal 2 minutes, abandoning supernatant reclaims and obtains protonema; A large amount of protonemas in the fermentor tank are collected with 4-8 layer filtered through gauze and are obtained.
The present invention has advantages such as the protonema development degree that the preparation method is easy, cost is low, obtain-cause.
Embodiment:
Below be embodiments of the invention, but content of the present invention is not limited thereto.
Embodiment 1:
1. the sterilization of ripe spore:
Select the sporangium of general living wall moss mature and plump, from the following 1cm of sporangium, the seta middle portion is cut off, and cleans the sporangium surface with clear water, uses 75% alcohol-pickled sporangium then, the tissue surface 0.5 minute of sterilizing in advance; Be that the mercury chloride of 1.0g/L and the solution that contains 1.0% meal lotion carry out disinfection to material sporangium surface with concentration then, the treatment time is 3 minutes; With aqua sterilisa soaking and washing sporangium 4 times repeatedly, the mercury chloride and the washing composition on cleaning material sporangium surface are residual again; With the fixing seta of aseptic nipper,, expose inner spore then, extrude spore in the centrifuge tube that fills sterilized water, make the spore dispersion suspension with tweezers with aseptic eye scissors transversely cutting sporangium top; The centrifuge tube good seal of spore will be filled, 40 ℃ water-bath moderate stimulations 40 seconds;
2. the sprouting on the solid medium:
Under aseptic condition, get density 10
4The spore of/ml is inoculated into and has added 2,4 dichloro benzene ethoxyacetic acid 2mg/L, water glass 2.0g/L, and in the MS substratum of sucrose 2.0%, pH5.8, culture temperature is 25 ± 3 ℃, illuminance is 4000lx, obtains the nascent bioblast by spore germination after 10 days.
3. a large amount of breedings and the collection of protonema
A large amount of breedings of protonema and the operation of collecting by centrifugal purification behind the shake-flask culture obtain.Specific as follows:
(1) a large amount of breedings of protonema
With tweezers gently the gripping volume be 10mm
3The protonema of sprouting, be inoculated into and added 2,4-D 2mg/L, water glass 2.0g/L, the volume of the MS liquid nutrient medium of sucrose 2.0%, pH5.8 is in the 50ml triangular flask, and the nutrient solution that adds in each triangular flask is a triangular flask volumetrical 1/3, and the 120rpm shaking table was cultivated 7 days, envrionment temperature is controlled at 25 ± 3 ℃, and light source is a diffused light; Carry out rolling bottle with identical substratum and control condition subsequently and cultivate, obtain a large amount of protonemas after 7 days.
(2) collection of protonema
Whole suspensions in the culturing bottle are poured in the centrifuge tube, 800rpm/min (rev/min) centrifugal 2 minutes, abandoning supernatant promptly reclaims and obtains a large amount of protonemas.
The protonema of above-mentioned centrifugal collection is cleaned with distilled water, be prepared into the simple and easy water load of liver moss protonema, be used for student's educational observation.
Embodiment 2:
Substantially with embodiment 1, difference is:
1. during the sterilization of ripe spore, 75% alcohol-pickled sporangium, the tissue surface 1 minute of sterilizing in advance, mercury chloride sterilization 5 minutes, the water-bath stimulation time is 50 seconds.
2. during the sprouting on the solid medium, spore density is 10
6/ ml, 2,4-D concentration is 5mg/L.
3. in a large amount of breedings of protonema and the collection:
With tweezers gently the gripping volume be 200mm
3Protonema, be inoculated into and added 2,4-D2.5mg/L, water glass 2.0g/L, the volume of the MS liquid nutrient medium of sucrose 2.0%, pH5.8 is in the 500ml triangular flask, and the nutrient solution that adds in each triangular flask is a triangular flask volumetrical 1/3, and the 120rpm shaking table was cultivated 7 days, envrionment temperature is controlled at 24 ℃, and light source is a diffused light; Carry out rolling bottle with identical substratum and control condition subsequently and cultivate, obtain a large amount of protonemas after 12 days.
Protonema in these culturing bottles all is used for the rolling bottle expanding propagation, under aseptic condition, all pour protonema in the 500ml triangle culturing bottle and nutrient solution thereof into the glass fermentor tank, add the 1000ml fresh medium, the bottom feeds sterile air (300L/ hour), make the protonema in the nutrient solution constantly seethe, cultivate and stop air inlet after 15 days, treat protonema sink to jar at the bottom of after, the nutrient solution that slowly will be in tank body top with peristaltic pump under aseptic condition extracts 2/3 out, add fresh sterile nutrient solution 5000ml again, continue to repeat enlarged culturing, repeat above-mentioned steps after 15 days, add the fresh sterile nutrient solution till the maximum useful volume 10L of tank body, 8 all pour out object in the jar after big, with obtaining a large amount of protonemas after pure 6 layers of filtered through gauze cleaning, after its 75% alcohol milling and extracting, be used to prepare antiviral and animal food refusal preparation with 20 times of volumes.
Embodiment 3:
Substantially with embodiment 1, difference is:
1. during the sterilization of ripe spore, 75% alcohol-pickled sporangium, the tissue surface 1.5 minutes of sterilizing in advance, mercury chloride sterilization 3 minutes, the water-bath stimulation time is 45 seconds.
2. during the sprouting on the solid medium, spore density is 10
5/ ml, 2,4-D concentration is 5mg/L.
3. in a large amount of breedings of protonema and the collection:
With tweezers gently the gripping volume be 1000mm
3Protonema, be inoculated into and added 2,4-D3mg/L, water glass 2.0g/L, the volume of the MS liquid nutrient medium of sucrose 2.0%, pH5.8 is in the 1000ml triangular flask, and the nutrient solution that adds in each triangular flask is a triangular flask volumetrical 1/3, and the 120rpm shaking table was cultivated 7 days, envrionment temperature is controlled at 24 ℃, and light source is a diffused light; Carry out rolling bottle with identical substratum and control condition subsequently and cultivate, 12 backs obtain a large amount of protonemas, can be used as the research material that liver moss grows.
Claims (1)
1. method of cultivating the general living wall moss protonema of breeding with cell engineering, the concrete steps that it is characterized in that breeding general living wall moss protonema are as follows:
A. the sterilization of ripe spore:
Select the sporangium of general living wall moss mature and plump, from the following 1-2cm of sporangium, the seta middle portion is cut off, and cleans the sporangium surface with clear water, uses 75% alcohol-pickled sporangium then, sterilizes in advance tissue surface 0.5-1 minute; Then the solution with mercury chloride that contains 1.0g/L and 1.0% meal lotion carries out disinfection to the sporangium surface, and the treatment time is 3-5 minute; With aqua sterilisa soaking and washing sporangium 4 times repeatedly, the mercury chloride on cleaning material sporangium surface and meal lotion are residual again; With the fixing seta of aseptic nipper,, expose inner spore then, extrude spore in the centrifuge tube that fills sterilized water, make the spore dispersion suspension with tweezers with aseptic eye scissors transversely cutting sporangium top; The centrifuge tube good seal of spore will be filled, 40 ℃ water-bath moderate stimulation 40-50 second;
B. the sprouting on the solid medium:
Under aseptic condition, getting the density that is obtained by step a is 10
4-10
6The spore of/ml is inoculated into and has added 2,4 dichloro benzene ethoxyacetic acid 2-5mg/L, water glass 2.0g/L, in the MS substratum of sucrose 2.0%, pH5.8, culture temperature is 25 ℃ ± 3 ℃, illuminance is 4000lx, obtains the primary protonema by spore germination after 10-20 days;
C. a large amount of breedings and the collection of protonema:
Is 10-1000mm with the tweezers gripping by the volume that the b step obtains
3The protonema of sprouting, be inoculated into and added 2,4-D 2-5mg/L, water glass 2.0g/L, the volume of the MS liquid nutrient medium of sucrose 2.0%, pH5.8 is in the 50-1000ml triangular flask, and the nutrient solution that adds in each triangular flask is triangular flask volumetrical 1/3-1/2, and the 120rpm shaking table was cultivated 7-10 days, envrionment temperature is controlled at 25 ± 3 ℃, and light source is a diffused light; Carry out the rolling bottle cultivation with identical substratum and control condition subsequently and obtain a large amount of protonemas after 7-15 days; As the more substantial protonema of needs, then under aseptic condition, the protonema that cultivation obtains is poured in the glass fermentor tank together with nutrient solution, the fresh MS nutrient solution that adds 3-10 times of volume, the bottom is pressed and was fed sterile air in 200L/ hour, make the protonema in the nutrient solution constantly seethe, with the no permanent sedimentary protonema in fermentor tank bottom is standard, cultivate and stop air inlet after 7-15 days, treat protonema sink to jar at the bottom of after, the nutrient solution that slowly will be in tank body top with peristaltic pump under aseptic condition extracts 2/3 out, add extremely originally 5 times of volume of fresh MS nutrient solutions again, continue to repeat enlarged culturing, till the tank body maximum volume, promptly obtain more substantial protonema so repeatedly; When collecting protonema, the whole suspensions of the protonema of cultivating in the triangular flask are poured in the centrifuge tube, used centrifugal 2 minutes of 800rpm, obtain protonema after the abandoning supernatant; A large amount of protonemas in the fermentor tank are collected with 4-8 layer filtered through gauze and are obtained.
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| CN109089882B (en) * | 2018-08-30 | 2022-02-08 | 云南省农业科学院花卉研究所 | Moss tissue culture and seedling culture method directly induced by spores |
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