CN101248759B - Artificial enlarge method (of) Rhodobryum giganteum gametophyte - Google Patents

Artificial enlarge method (of) Rhodobryum giganteum gametophyte Download PDF

Info

Publication number
CN101248759B
CN101248759B CN2008100346502A CN200810034650A CN101248759B CN 101248759 B CN101248759 B CN 101248759B CN 2008100346502 A CN2008100346502 A CN 2008100346502A CN 200810034650 A CN200810034650 A CN 200810034650A CN 101248759 B CN101248759 B CN 101248759B
Authority
CN
China
Prior art keywords
protonema
medium
days
gametophyte
gametophytic
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired - Fee Related
Application number
CN2008100346502A
Other languages
Chinese (zh)
Other versions
CN101248759A (en
Inventor
娄玉霞
陈圆圆
曹同
郭水良
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shanghai Normal University
University of Shanghai for Science and Technology
Original Assignee
Shanghai Normal University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Shanghai Normal University filed Critical Shanghai Normal University
Priority to CN2008100346502A priority Critical patent/CN101248759B/en
Publication of CN101248759A publication Critical patent/CN101248759A/en
Application granted granted Critical
Publication of CN101248759B publication Critical patent/CN101248759B/en
Expired - Fee Related legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Landscapes

  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

The invention discloses a method of manual extensive-reproduction of rhodobryum giganteum gametocyte, which includes steps in the following order: (1) sterilizing the surface of the rhodobryum giganteum gametocyte which grows in a natural environment with unexpanded leaf blades; cutting the stem tip and inoculating to the culture medium of inducing the gametocyte to generate the protonema and culturing for 40-60 days; (2) transplanting the protonema obtained in step (1) to the extensive-reproduction protonema culture medium and culturing for 40-60 days; (3) transplanting the extensive reproduced protonema in step (2) to the culture medium of inducing the protonema to generate the gametocyte and culturing for 40-60 days. The invention initiates the development of the method of manual extensive-reproduction of rhodobryum giganteum gametocyte and bridges the gap of the prior art. The manual extensive reproduction not only greatly facilitates the production of high intensification and high density industrialization, but also facilitates automatization of production control; which achieves the mass production of manual extensive-reproduction of rhodobryum giganteum in a short time and provides a market guarantee for the pharmacy field with significant economic benefits.

Description

The gametophytic method of a kind of artificial expansion numerous warm ground Da Ye moss
Technical field
The present invention relates to the gametophytic method of a kind of artificial expansion numerous warm ground Da Ye moss, belong to technical field of bioengineering.
Background technology
Warm ground Da Ye moss (Rhodobryum giganteum) is subordinate to Bryaceae rhodobryum spp plant, and (Rhodobryum roseum) is referred to as rose rhodobryum herb with congener Da Ye moss.Rose rhodobryum herb is that medicine is commonly used in the place, also is the ethnic drug that Yi nationality, the Dai Nationality, Hani etc. use.Its curative effect in treatment coronary heart disease, angina pectoris and diseases such as hypertension and high fat of blood has all been determined in the relevant rhodobryum spp plant of China (serving as for the examination material with warm ground Da Ye moss mainly) Pharmacological action study, modern clinical research and preparation application study in recent years.This kind medicinal plant preparation has the rhodobryum roseum tablet of Yunnan Ying Mao Biology Pharmacy Co., Ltd production and rose rhodobryum herb tablet and the injection that Changwu of Shaanxi pharmaceutical factory produces at present.
The rose rhodobryum herb that uses as medicinal material-warm ground Da Ye moss all derives from wild resource at present, with all herbal medicine, gathers in summer, autumn, and it mainly is the gametophyte of drying shrinkage that medicinal material is formed.Utilize wild warm ground Da Ye moss to carry out extensive pharmaceutical manufacturing and will face problems: at first, often be subjected to the influence that natural environment changes in the self-sow process, can there be various variation individualities or colony, can influence the uniformity and the stability of resource output and resource proterties; Secondly, the stability that all can not influence quality of medicinal material on an equal basis in the difference of picking time, the place of production; The more important thing is that wild plant resource is scattered in the distribution of occurring in nature, almost there is not large tracts of land phenomenon in blocks, in case wild plant becomes important exploiting natural resources, only depend on wild resource to be difficult to satisfy market demand on the one hand, on the other hand, under the pressure of development and use, very easily destroyed, existence constitutes directly threat to species.Therefore, by the numerous warm ground of artificial expansion Da Ye moss, for pharmaceutical field provides abundant vegetable drug imperative.But also do not expand numerous gametophytic culture technique in vitro in a large number in the prior art at present, more the rhodobryum spp Plant Tissue Breeding is not become gametophytic correlation technique report about the rhodobryum spp plant.
Summary of the invention
The object of the present invention is to provide the gametophytic method of a kind of artificial expansion numerous warm ground Da Ye moss, to fill up the blank of prior art, realize the numerous warm ground of artificial a large amount of expansions Da Ye moss, the purpose of enriching vegetable drug is provided for pharmaceutical field.
For achieving the above object, the technical solution used in the present invention is as follows:
The gametophytic method of artificial expansion disclosed by the invention numerous warm ground Da Ye moss comprises following sequential steps:
1) will grow in the warm ground Da Ye moss gametophyte surface sterilization that does not launch blade in the natural environment after, cutting stem apex is inoculated on the medium of inducing gametophyte generation protonema, cultivated 40～60 days, condition of culture is: 20～25 ℃ of temperature, intensity of illumination 2000～4000lx, light application time 10～12 hours/day; Described culture medium prescription of inducing gametophyte to produce protonema is: improvement Knop medium+0～2.0mg/l2,4-dichlorphenoxyacetic acid+0～0.10mg/l 6-benzylaminopurine+5～10g/l sucrose; Wherein improveing the Knop culture medium prescription is: KCl 76.04mg/l, MgSO 47H 2O 828.2mg/l, KH 2PO 4250.4mg/l, Ca (NO 3) 24H 2O 1001.3mg/l, CuSO 45H 2O 0.027mg/l, KI0.014mg/l, ZnSO 47H 2O 0.027mg/l, H 3BO 30.309mg/l, Na 2MoO 42H 2O 0.012mg/l, MnCl 24H 2O 0.198mg/l, CoCl 26H 2O 0.027mg/l, FeSO 47H 2O 27.8mg/l, Na 2-EDTA 37.3mg/l, tartaric acid ammonia 0.1151mg/l;
2) protonema that step 1) is obtained changes on the protonema expansion breeding culture medium, cultivates 40～60 days, and condition of culture is: 20～25 ℃ of temperature, intensity of illumination 2000～4000lx, light application time 10～12 hours/day; The prescription that described protonema expands breeding culture medium is: improvement Knop medium+0～2.0mg/l 2,4 dichlorophenoxyacetic acid or 0～2.0mg/l 6-furfuryl group aminopurine+5～10g/l sucrose, and wherein improve the Knop culture medium prescription and be: KCl 76.04mg/l, MgSO 47H 2O 828.2mg/l, KH 2PO 4250.4mg/l, Ca (NO 3) 24H 2O 1001.3mg/l, CuSO 45H 2O 0.027mg/l, KI0.014mg/l, ZnSO 47H 2O0.027mg/l, H 3BO 30.309mg/l, Na 2MoO 42H 2O 0.012mg/l, MnCl 24H 2O0.198mg/l, CoCl 26H 2O 0.027mg/l, FeSO 47H 2O 27.8mg/l, Na 2-EDTA 37.3mg/l, tartaric acid ammonia 0.115lmg/l;
3) with step 2) expand numerous protonema that obtains and change over to and induce protonema to form on the gametophytic medium, to cultivate 40～60 days, condition of culture is: 25～30 ℃ of temperature, intensity of illumination 3000～4000lx, light application time 12～14 hours/day; Describedly induce protonema to form gametophytic culture medium prescription to be: improvement White medium+0～2.0mg/l 6-furfuryl group aminopurine+5～10g/l sucrose, wherein improve the White culture medium prescription and be: KNO 380mg/l, MgSO 47H 2O 720mg/l, NaH 2PO 4H 2O 16.5mg/l, KCl 65mg/l, Na 2SO 4200mg/l, Ca (NO 3) 24H 2O 300mg/l, CuSO 45H 2O 0.001mg/l, ZnSO 47H 2O 3.0mg/l, H 3BO 31.5mg/l, Na 2MoO 42H 2O 0.0001mg/l, MnSO 44H 2O7.0mg/l, FeNa-EDTA 4.588mg/l, inositol 100mg/l, nicotinic acid 0.3mg/l, thiamine hydrochloride 0.1mg/l, pyridoxine hydrochloride 0.1mg/l, glycine 3mg/l.
Be the alcoholic solution of 70～75% volume fractions and the mercuric chloride solution of 0.1%～0.2% mass fraction to gametophyte surface disinfector for disinfecting in the described method.
The stem apex that cuts in the described method derives from the gametophyte that spire does not launch as yet, and length is 0.5～1.0cm.
Described culture medium prescription of inducing gametophyte to produce protonema is preferably: improvement Knop+2.0mg/l2,4-dichlorphenoxyacetic acid+0.1mg/l 6-benzylaminopurine+5～10g/l sucrose.
The prescription that described protonema expands breeding culture medium is preferably: improvement Knop medium+1.0mg/l 2,4 dichlorophenoxyacetic acid or 1.0mg/l 6-furfuryl group aminopurine+5～10g/l sucrose.
Describedly induce protonema to form gametophytic culture medium prescription to be preferably: improvement White medium+1.0mg/l 6-furfuryl group aminopurine or additional any plant hormone+5～10g/l sucrose.
Condition of culture in the described step 1) is preferably: 23 ± 2 ℃ of temperature, and intensity of illumination 2500 ± 250lx, light application time 10 hours/day was cultivated 50 days.
Described step 2) condition of culture in is preferably: 23 ± 2 ℃ of temperature, and intensity of illumination 3000 ± 250lx, light application time 12 hours/day was cultivated 55 days.
Condition of culture in the described step 3) is preferably: 27 ± 2 ℃ of temperature, and intensity of illumination 3500 ± 250lx, light application time 14 hours/day was cultivated 60 days.
Beneficial effect of the present invention is: set up the gametophytic method of the numerous warm ground of artificial expansion Da Ye moss first, filled up the blank of prior art; And, using artificial expanding propagation method of the present invention, the utmost point is beneficial to highly intensification and the production of producing of high density worker, also is beneficial to automation control and produces; Can realize artificial a large amount of numerous warm ground Da Ye moss of expanding at short notice, ensure that for pharmaceutical field provides market economic benefit is obvious.
Embodiment
The gametophytic method of artificial expansion disclosed by the invention numerous warm ground Da Ye moss comprises following sequential steps:
1) will grow in the warm ground Da Ye moss gametophyte surface sterilization that does not launch blade in the natural environment after, cutting stem apex is inoculated on the medium of inducing gametophyte generation protonema, cultivated 40～60 days, condition of culture is: 20～25 ℃ of temperature, intensity of illumination 2000～4000lx, light application time 10～12 hours/day; Described culture medium prescription of inducing gametophyte to produce protonema is: improvement Knop medium+0～2.0mg/l2,4-dichlorphenoxyacetic acid+0～0.10mg/l 6-benzylaminopurine+5～10g/l sucrose; Wherein improveing the Knop culture medium prescription is: KCl 76.04mg/l, MgSO 47H 2O 828.2mg/l, KH 2PO 4250.4mg/l, Ca (NO 3) 24H 2O 1001.3mg/l, CuSO 45H 2O 0.027mg/l, KI0.014mg/l, ZnSO 47H 2O 0.027mg/l, H 3BO 30.309mg/l, Na 2MoO 42H 2O 0.012mg/l, MnCl 24H 2O 0.198mg/l, CoCl 26H 2O 0.027mg/l, FeSO 47H 2O 27.8mg/l, Na 2-EDTA 37.3mg/l, tartaric acid ammonia 0.1151mg/l;
2) protonema that step 1) is obtained changes on the protonema expansion breeding culture medium, cultivates 40～60 days, and condition of culture is: 20～25 ℃ of temperature, intensity of illumination 2000～4000lx, light application time 10～12 hours/day; The prescription that described protonema expands breeding culture medium is: improvement Knop medium+0～2.0mg/l 2,4 dichlorophenoxyacetic acid or 0～2.0mg/l 6-furfuryl group aminopurine+5～10g/l sucrose, and wherein improve the Knop culture medium prescription and be: KCl 76.04mg/l, MgSO 47H 2O 828.2mg/l, KH 2PO 4250.4mg/l, Ca (NO 3) 24H 2O 1001.3mg/l, CuSO 45H 2O 0.027mg/l, KI0.014mg/l, ZnSO 47H 2O0.027mg/l, H 3BO 30.309mg/l, Na 2MoO 42H 2O 0.012mg/l, MnCl 24H 2O0.198mg/l, CoCl 26H 2O 0.027mg/l, FeSO 47H 2O 27.8mg/l, Na 2-EDTA 37.3mg/l, tartaric acid ammonia 0.1151mg/l;
3) with step 2) expand numerous protonema that obtains and change over to and induce protonema to form on the gametophytic medium, to cultivate 40～60 days, condition of culture is: 25～30 ℃ of temperature, intensity of illumination 3000～4000lx, light application time 12～14 hours/day; Describedly induce protonema to form gametophytic culture medium prescription to be: improvement White medium+0～2.0mg/l 6-furfuryl group aminopurine+5～10g/l sucrose, wherein improve the White culture medium prescription and be: KNO 380mg/l, MgSO 47H 2O 720mg/l, NaH 2PO 4H 2O 16.5mg/l, KCl 65mg/l, Na 2SO 4200mg/l, Ca (NO 3) 24H 2O 300mg/l, CuSO 45H 2O 0.001mg/l, ZnSO 47H 2O 3.0mg/l, H 3BO 31.5mg/l, Na2MoO 42H 2O 0.0001mg/l, MnSO 44H 2O7.0mg/l, FeNa-EDTA 4.588mg/l, inositol 100mg/l, nicotinic acid 0.3mg/l, thiamine hydrochloride 0.1mg/l, pyridoxine hydrochloride 0.1mg/l, glycine 3mg/l.
Be the alcoholic solution of 70～75% volume fractions and the mercuric chloride solution of 0.1%～0.2% mass fraction to gametophyte surface disinfector for disinfecting in the described method.
The stem apex that cuts in the described method derives from the gametophyte that spire does not launch as yet, and length is 0.5～1.0cm.
Below in conjunction with embodiment the present invention is done further detailed, complete explanation:
Embodiment 1
Carry out surface sterilization with growing in the warm ground Da Ye moss gametophyte that does not launch blade in the natural environment, disinfectant is the alcoholic solution of 70～75% volume fractions and the mercuric chloride solution of 0.1%～0.2% mass fraction; After the sterilization, be that the stem apex of 0.5～1.0cm is inoculated into and induces gametophyte to produce on the medium of protonema with cutting length, condition of culture is: 23 ± 2 ℃ of temperature, and intensity of illumination 2500 ± 250lx, light application time 10 hours/day was cultivated 50 days.
Preparation induces gametophyte to produce the medium of protonema according to a conventional method, and prescription consists of following five kinds:
A) improvement Knop+5～10g/l sucrose;
B) improvement Knop+0.1mg/l 6-benzylaminopurine+5～10g/l sucrose;
C) improvement Knop+2.0mg/l 2,4 dichlorophenoxyacetic acid+5～10g/l sucrose;
D) improvement Knop+1.0mg/l 2,4 dichlorophenoxyacetic acid+0.05mg/l 6-benzylaminopurine+5～10g/l sucrose;
E) improvement Knop+2.0mg/l 2,4 dichlorophenoxyacetic acid+0.1mg/l 6-benzylaminopurine+5～10g/l sucrose.
Described improvement Knop culture medium prescription is: KCl 76.04mg/l, MgSO 47H 2O 828.2mg/l, KH 2PO 4250.4mg/l, Ca (NO 3) 24H 2O 1001.3mg/l, CuSO 45H 2O 0.027mg/l, KI0.014mg/l, ZnSO 47H 2O 0.027mg/l, H 3BO 30.309mg/l, Na 2MoO 42H 2O0.012mg/l, MnCl 24H 2O 0.198mg/l, CoCl 26H 2O 0.027mg/l, FeSO 47H 2O27.8mg/l, Na 2-EDTA 37.3mg/l, tartaric acid ammonia 0.1151mg/l.
Cultivation results is as follows:
A) in the medium of improvement Knop+5～10g/l sucrose, explant 100% brown stain death fails to induce the formation protonema;
B) in the medium of improvement Knop+0.1mg/l 6-benzylaminopurine+5～10g/l sucrose, have on 8.2% the explant to have produced single gametophyte budlet, all the other whole brown stain death;
C) improvement Knop+2.0mg/l 2, in the medium of 4-dichlorphenoxyacetic acid+5～10g/l sucrose, only there is 11.6% explant to produce protonema, protonema is green, by explant and the contacted position of medium to around be radial growth, be 0.92cm through measuring its growth circle average diameter;
D) improvement Knop+1.0mg/l 2, in the medium of 4-dichlorphenoxyacetic acid+0.05mg/l 6-benzylaminopurine+5～10g/l sucrose, there is 38.6% explant to produce protonema, protonema is green, by explant and the contacted position of medium to around be radial growth, be 1.12cm through measuring its growth circle average diameter;
E) improvement Knop+2.0mg/l 2, in the medium of 4-dichlorphenoxyacetic acid+0.1mg/l 6-benzylaminopurine+5～10g/l sucrose, there is 88.6% explant to produce protonema, protonema is green, by explant and the contacted position of medium to around be radial growth, be 4.62cm through measuring its growth circle average diameter.
More above-mentioned cultivation results is as can be seen: under the isolated condition, induce warm ground Da Ye moss gametophyte to form protonema, the interpolation of hormone is necessary in the medium.On the basis of improvement Knop medium, the kind and the concentration thereof of adding hormone have material impact to inducing gametophyte to form protonema.Single interpolation 6-benzylaminopurine and 2, the 4-dichlorphenoxyacetic acid all is unfavorable for the formation of protonema, and induce effect obvious during the two compound use, induce effect best when especially cooperating 0.1mg/l 6-benzylaminopurine with the 2.0mg/l 2,4 dichlorophenoxyacetic acid.
Embodiment 2
Carry out surface sterilization with growing in the warm ground Da Ye moss gametophyte that does not launch blade in the natural environment, disinfectant is the alcoholic solution of 70～75% volume fractions and the mercuric chloride solution of 0.1%～0.2% mass fraction; After the sterilization, with cutting length is that the stem apex of 0.5～1.0cm is inoculated into and induces gametophyte to produce on the medium of protonema, described culture medium prescription of inducing gametophyte to produce protonema is: improvement Knop+2.0mg/l 2,4-dichlorphenoxyacetic acid+0.1mg/l 6-benzylaminopurine+5～10g/l sucrose is wherein improved the Knop culture medium prescription and is: KCl 76.04mg/l, MgSO 47H 2O 828.2mg/l, KH 2PO 4250.4mg/l, Ca (NO 3) 24H 2O 1001.3mg/l, CuSO 45H 2O 0.027mg/l, KI0.014mg/l, ZnSO 47H 2O0.027mg/l, H 3BO 30.309mg/l, Na 2MoO 42H 2O 0.012mg/l, MnCl 24H 2O0.198mg/l, CoCl 26H 2O 0.027mg/l, FeSO 47H 2O 27.8mg/l, Na 2-EDTA 37.3mg/l, tartaric acid ammonia 0.1151mg/l are prepared into the medium of inducing gametophyte to produce protonema according to a conventional method, cultivate by following five kinds of condition of culture:
A) temperature is 23 ± 2 ℃, intensity of illumination 2500 ± 250lx, and light application time 10 hours/day was cultivated 50 days;
B) temperature is 23 ± 2 ℃, intensity of illumination 2500 ± 250lx, and light application time 12 hours/day was cultivated 50 days;
C) temperature is 23 ± 2 ℃, intensity of illumination 2500 ± 250lx, and light application time 10 hours/day was cultivated 40 days;
D) temperature is 23 ± 2 ℃, intensity of illumination 2500 ± 250lx, and light application time 10 hours/day was cultivated 60 days;
E) temperature is 23 ± 2 ℃, intensity of illumination 3500 ± 250lx, and light application time 10 hours/day was cultivated 50 days.
Cultivation results is as follows:
A) temperature is 23 ± 2 ℃, intensity of illumination 2500 ± 250lx, light application time 10 hours/day, when cultivating 50 days, there is 88.6% explant to produce protonema, protonema is green, by explant and the contacted position of medium to around be radial growth, grow through measuring it that to enclose average diameter be 4.62cm;
B) temperature is 23 ± 2 ℃, intensity of illumination 2500 ± 250lx, light application time 12 hours/day, when cultivating 50 days, there is 68.4% explant to produce protonema, protonema is green, by explant and the contacted position of medium to around be radial growth, grow through measuring it that to enclose average diameter be 2.62cm;
C) temperature is 23 ± 2 ℃, intensity of illumination 2500 ± 250lx, light application time 10 hours/day, when cultivating 40 days, there is 86.4% explant to produce protonema, protonema is green, by explant and the contacted position of medium to around be radial growth, grow through measuring it that to enclose average diameter be 3.38cm;
D) temperature is 23 ± 2 ℃, intensity of illumination 2500 ± 250lx, light application time 10 hours/day, when cultivating 60 days, have 88.6% explant to produce protonema, protonema by explant and the contacted position of medium to around be radial growth, color becomes dirty-green by original green, especially the obvious blackening in centre of protonema growth circle is 4.66cm through measuring its growth circle average diameter;
E) temperature is 23 ± 2 ℃, intensity of illumination 3500 ± 250lx, light application time 10 hours/day, when cultivating 50 days, there is 48.2% explant to produce protonema, protonema is green, by explant and the contacted position of medium to around be radial growth, grow through measuring it that to enclose average diameter be 2.34cm.
More above-mentioned cultivation results as can be seen, intensity of illumination and light application time are influential for inducing explant to produce protonema, illumination enhancing and light application time prolong the formation that is unfavorable for inducing protonema.In addition, incubation time is influential to the amount of growth of the protonema of inducing formation, and incubation time prolongs, and the protonema amount of growth increases.But incubation time is long, can cause the protonema growth retardation because of the deficiency of nutrient in the medium and moisture, even dead.
Embodiment 3
The green protonema that embodiment 1 is obtained changes on the protonema expansion breeding culture medium, and condition of culture is: 23 ± 2 ℃ of temperature, and intensity of illumination 3000 ± 250lx, light application time 12 hours/day was cultivated 55 days.
Prepare protonema according to a conventional method and expand breeding culture medium, prescription consists of following four kinds:
A) improvement Knop medium+1.0mg/l 2,4 dichlorophenoxyacetic acid+5～10g/l sucrose;
B) improvement Knop medium+2.0mg/l 2,4 dichlorophenoxyacetic acid+5～10g/l sucrose;
C) improvement Knop medium+1.0mg/l 6-furfuryl group aminopurine+5～10g/l sucrose;
D) improvement Knop medium+2.0mg/l 6-furfuryl group aminopurine+5～10g/l sucrose.
Described improvement Knop culture medium prescription is: KCl 76.04mg/l, MgSO 47H 2O 828.2mg/l, KH 2PO 4250.4mg/l, Ca (NO 3) 24H 2O 1001.3mg/l, CuSO 45H 2O 0.027mg/l, KI0.014mg/l, ZnSO 47H 2O 0.027mg/l, H 3BO 30.309mg/l, Na 2MoO 42H 2O0.012mg/l, MnCl 24H 2O 0.198mg/l, CoCl 26H 2O 0.027mg/l, FeSO 47H 2O27.8mg/l, Na 2-EDTA 37.3mg/l, tartaric acid ammonia 0.1151mg/l.
Cultivation results is as follows:
A) improvement Knop medium+1.0mg/l 2, in the medium of 4-dichlorphenoxyacetic acid+5～10g/l sucrose, a small amount of protonema of the about 3～4mm of diameter of initial inoculation is to growth radially all around, form circular growth circle, growth rate is very fast, almost covering with media surface, is 6.52cm through measuring its growth circle average diameter, and protonema is green;
B) improvement Knop medium+2.0mg/l 2, in the medium of 4-dichlorphenoxyacetic acid+5～10g/l sucrose, a small amount of protonema of the about 3～4mm of diameter of initial inoculation is to growth radially all around, form circular growth circle, growth rate is slower, through measuring its growth circle average diameter is 3.26cm, and protonema is green;
C) in the medium of improvement Knop medium+1.0mg/l 6-furfuryl group aminopurine+5～10g/l sucrose, a small amount of protonema of the about 3～4mm of diameter of initial inoculation is to growth radially all around, form circular growth circle, growth rate is very fast, almost cover with media surface, through measuring its growth circle average diameter is 6.36cm, and protonema is green, and accidental gametophyte forms;
D) in the medium of improvement Knop medium+2.0mg/l 6-furfuryl group aminopurine+5～10g/l sucrose, a small amount of protonema of the about 3～4mm of diameter of initial inoculation is to growth radially all around, form circular growth circle, growth rate is slower, through measuring its growth circle average diameter is 4.18cm, and protonema is green.
More above-mentioned cultivation results as can be seen, on the basis of improvement Knop medium, the difference of adding hormone kind and concentration has a significant impact the growth of protonema.2,4 dichlorophenoxyacetic acid and 6-furfuryl group aminopurine all help the growth of protonema, but the too high growth that is unfavorable for protonema of hormone concentration.
Embodiment 4
The green protonema that embodiment 1 is obtained changes on the protonema expansion breeding culture medium, the prescription that described protonema expands breeding culture medium is: improvement Knop medium+1.0mg/l 2,4-dichlorphenoxyacetic acid+5～10g/l sucrose is wherein improved the Knop culture medium prescription and is: KCl 76.04mg/l, MgSO 47H 2O 828.2mg/l, KH 2PO 4250.4mg/l, Ca (NO 3) 24H 2O 1001.3mg/l, CuSO 45H 2O 0.027mg/l, KI0.014mg/l, ZnSO 47H 2O 0.027mg/l, H 3BO 30.309mg/l, Na 2MoO 42H 2O0.012mg/l, MnCl 24H 2O 0.198mg/l, CoCl 26H 2O 0.027mg/l, FeSO 47H 2O27.8mg/l, Na 2-EDTA 37.3mg/l, tartaric acid ammonia 0.1151mg/l are prepared into protonema according to a conventional method and expand breeding culture medium, cultivate by following five kinds of condition of culture:
A) temperature is 23 ± 2 ℃, intensity of illumination 3000 ± 250lx, and light application time 10 hours/day was cultivated 55 days;
B) temperature is 23 ± 2 ℃, intensity of illumination 3000 ± 250lx, and light application time 12 hours/day was cultivated 55 days;
C) temperature is 23 ± 2 ℃, intensity of illumination 3000 ± 250lx, and light application time 12 hours/day was cultivated 40 days;
D) temperature is 23 ± 2 ℃, intensity of illumination 3000 ± 250lx, and light application time 12 hours/day was cultivated 60 days;
E) temperature is 23 ± 2 ℃, intensity of illumination 2500 ± 250lx, and light application time 12 hours/day was cultivated 55 days.
Cultivation results is as follows:
A) temperature is 23 ± 2 ℃, intensity of illumination 3000 ± 250lx, light application time 10 hours/day, when cultivating 55 days, a small amount of protonema of the about 3～4mm of diameter of initial inoculation forms circular growth circle to growth radially all around, through measuring its growth circle average diameter is 4.62cm, and protonema is green;
B) temperature is 23 ± 2 ℃, intensity of illumination 3000 ± 250lx, light application time 12 hours/day, when cultivating 55 days, a small amount of protonema of the about 3～4mm of diameter of initial inoculation forms circular growth circle to growth radially all around, and protonema almost covers with media surface, through measuring its growth circle average diameter is 6.52cm, and protonema is green;
C) temperature is 23 ± 2 ℃, intensity of illumination 3000 ± 250lx, light application time 12 hours/day, when cultivating 40 days, a small amount of protonema of the about 3～4mm of diameter of initial inoculation forms circular growth circle to growth radially all around, through measuring its growth circle average diameter is 5.73cm, and protonema is green;
D) temperature is 23 ± 2 ℃, intensity of illumination 3000 ± 250lx, light application time 12 hours/day, when cultivating 60 days, a small amount of protonema of the about 3～4mm of diameter of initial inoculation forms circular growth circle to growth radially all around, through measuring its growth circle average diameter is 6.53cm, and protonema is green;
E) temperature is 23 ± 2 ℃, intensity of illumination 2500 ± 250lx, light application time 12 hours/day, when cultivating 55 days, a small amount of protonema of the about 3～4mm of diameter of initial inoculation forms circular growth circle to growth radially all around, through measuring its growth circle average diameter is 4.58cm, and protonema is green.
More above-mentioned cultivation results as can be seen, it is bigger that intensity of illumination and light application time expand the influence of numerous propagation to protonema.The too weak and too short growth that is unfavorable for warm ground Da Ye moss protonema of light application time of illumination.In addition, the length of incubation time is obvious to the amount of growth influence of protonema, and under the condition of nutrient and water supply abundance, incubation time is long more, and the amount of growth of protonema is big more.
Embodiment 5
The green protonema that embodiment 3 is obtained changes over to induces protonema to form on the gametophytic medium, and condition of culture is: 27 ± 2 ℃ of temperature, and intensity of illumination 3500 ± 250lx, light application time 14 hours/day was cultivated 60 days.
Preparation induces protonema to form gametophytic medium according to a conventional method, and prescription consists of following three kinds:
A) improvement White medium+1.0mg/l 6-furfuryl group aminopurine+5～10g/l sucrose;
B) improvement White medium+2.0mg/l 6-furfuryl group aminopurine+5～10g/l sucrose;
C) improvement White medium+5～10g/l sucrose.
Described improvement White culture medium prescription is: KNO 380mg/l, MgSO 47H 2O 720mg/l, NaH 2PO 4H 2O 16.5mg/l, KCl 65mg/l, Na 2SO 4200mg/l, Ca (NO 3) 24H 2O300mg/l, CuSO 45H 2O 0.001mg/l, ZnSO 47H 2O 3.0mg/l, H 3BO 31.5mg/l, Na 2MoO 42H 2O 0.0001mg/l, MnSO 44H 2O 7.0mg/l, FeNa-EDTA 4.588mg/l, inositol 100mg/l, nicotinic acid 0.3mg/l, thiamine hydrochloride 0.1mg/l, pyridoxine hydrochloride 0.1mg/l, glycine 3mg/l.
Cultivation results is as follows:
A) in the medium of improvement White medium+1.0mg/l 6-furfuryl group aminopurine+5～10g/l sucrose, formed a large amount of gametophytes, by statistics, newborn gametophyte number is 22 strains in average every bottle of medium, and average plant height is 1.82cm;
B) in improvement White medium+2.0mg/l 6-furfuryl group aminopurine+5～10g/l sucrose, formed more gametophyte, by statistics, newborn gametophyte number is 31 strains in average every bottle of medium, and average plant height is 1.01cm;
C) in improvement White medium+5～10g/l sucrose, formed more gametophyte, by statistics, newborn gametophyte number is 12 strains in average every bottle of medium, and average plant height is 2.06cm.
More above-mentioned cultivation results just can obtain a fairly large number of gametophyte as can be seen on improvement White medium.The 6-furfuryl group aminopurine that adds suitable concentration on the basis of improvement White medium can promote gametophytic being differentiated to form more, but is unfavorable for the gametophyte growth.
Embodiment 6
The green protonema that embodiment 3 is obtained changes over to induces protonema to form on the gametophytic medium, describedly induce protonema to form gametophytic culture medium prescription to be: improvement White medium+1.0mg/l 6-furfuryl group aminopurine+5～10g/l sucrose, wherein improve the White culture medium prescription and be: KNO 380mg/l, MgSO 47H 2O 720mg/l, NaH 2PO 4H 2O 16.5mg/l, KCl 65mg/l, Na 2SO 4200mg/l, Ca (NO 3) 24H 2O 300mg/l, CuSO 45H 2O 0.001mg/l, ZnSO 47H 2O 3.0mg/l, H 3BO 31.5mg/l, Na 2MoO 42H 2O 0.0001mg/l, MnSO 44H 2O 7.0mg/l, FeNa-EDTA4.588mg/l, inositol 100mg/l, nicotinic acid 0.3mg/l, thiamine hydrochloride 0.1mg/l, pyridoxine hydrochloride 0.1mg/l, glycine 3mg/l, be prepared into according to a conventional method and induce protonema to form gametophytic medium, cultivate by following five kinds of condition of culture:
A) temperature is 27 ± 2 ℃, intensity of illumination 3500 ± 250lx, and light application time 14 hours/day was cultivated 60 days;
B) temperature is 27 ± 2 ℃, intensity of illumination 3500 ± 250lx, and light application time 14 hours/day was cultivated 40 days;
C) temperature is 27 ± 2 ℃, intensity of illumination 3500 ± 250lx, and light application time 14 hours/day was cultivated 50 days;
D) temperature is 27 ± 2 ℃, intensity of illumination 3500 ± 250lx, and light application time 12 hours/day was cultivated 60 days;
E) temperature is 27 ± 2 ℃, intensity of illumination 2500 ± 250lx, and light application time 14 hours/day was cultivated 60 days.
Cultivation results is as follows:
A) temperature is 27 ± 2 ℃, intensity of illumination 3500 ± 250lx, and light application time 14 hours/day when cultivating 60 days, has been differentiated to form a large amount of gametophytes on the protonema, and by statistics, newborn gametophyte number is 22 strains in average every bottle of medium, and average plant height is 1.82cm;
B) temperature is 27 ± 2 ℃, intensity of illumination 3500 ± 250lx, and light application time 14 hours/day when cultivating 40 days, has been differentiated to form gametophyte on the protonema, and by statistics, newborn gametophyte number is 16 strains in average every bottle of medium, and average plant height is 1.04cm;
C) temperature is 27 ± 2 ℃, intensity of illumination 3500 ± 250lx, and light application time 14 hours/day when cultivating 50 days, has been differentiated to form gametophyte on the protonema, and by statistics, newborn gametophyte number is 22 strains in average every bottle of medium, and average plant height is 1.3cm;
D) temperature is 27 ± 2 ℃, intensity of illumination 3500 ± 250lx, and light application time 12 hours/day when cultivating 60 days, has been differentiated to form gametophyte on the protonema, and by statistics, newborn gametophyte number is 9 strains in average every bottle of medium, and average plant height is 1.06cm;
E) temperature is 27 ± 2 ℃, intensity of illumination 2500 ± 250lx, and light application time 14 hours/day when cultivating 60 days, has been differentiated to form gametophyte on the protonema, and by statistics, newborn gametophyte number is 8 strains in average every bottle of medium, and average plant height is 1.09cm.
More above-mentioned cultivation results as can be seen, it is bigger that intensity of illumination and light application time form influence to gametophyte.More weak and the light application time of illumination is short to be unfavorable for the gametophytic formation of warm ground Da Ye moss, and this is similar with a lot of requirements that is differentiated to form optical condition of other higher plant buds.In addition, the length of incubation time is obvious to gametophytic growth effect, and under the condition of nutrient and water supply abundance, incubation time is long more, and it is high more that gametophyte is grown.

Claims (9)

1. one kind is manually expanded the gametophytic method of numerous warm ground Da Ye moss, it is characterized in that described method comprises following sequential steps:
1) will grow in the warm ground Da Ye moss gametophyte surface sterilization that does not launch blade in the natural environment after, cutting stem apex is inoculated on the medium of inducing gametophyte generation protonema, cultivated 40～60 days, condition of culture is: 20～25 ℃ of temperature, intensity of illumination 2000～4000lx, light application time 10～12 hours/day; Described culture medium prescription of inducing gametophyte to produce protonema is:
Improvement Knop medium+1.0～2.0mg/l 2,4 dichlorophenoxyacetic acid+0.05～0.10mg/l 6-benzylaminopurine+5～10g/l sucrose;
Perhaps, improvement Knop medium+2.0mg/l 2,4 dichlorophenoxyacetic acid+5～10g/l sucrose;
Perhaps, improvement Knop medium+0.1mg/l 6-benzylaminopurine+5～10g/l sucrose;
More than induce gametophyte to produce that improvement Knop culture medium prescription is in the medium of protonema: KCl76.04mg/l, MgSO 47H 2O 828.2mg/l, KH 2PO 4250.4mg/l, Ca (NO 3) 24H 2O1001.3mg/l, CuSO 45H 2O 0.027mg/l, KI 0.014mg/l, ZnSO 47H 2O 0.027mg/l, H 3BO 30.309mg/l, Na 2MoO 42H 2O 0.012mg/l, MnCl 24H 2O 0.198mg/l, CoCl 26H 2O 0.027mg/l, FeSO 47H 2O 27.8mg/l, Na 2-EDTA 37.3mg/l, tartaric acid ammonia 0.1151mg/l;
2) protonema that step 1) is obtained changes on the protonema expansion breeding culture medium, cultivates 40～60 days, and condition of culture is: 20～25 ℃ of temperature, intensity of illumination 2000～4000lx, light application time 10～12 hours/day; The prescription that described protonema expands breeding culture medium is: improvement Knop medium+1.0～2.0mg/l2,4-dichlorphenoxyacetic acid or 6-furfuryl group aminopurine+5～10g/l sucrose are wherein improved the Knop culture medium prescription and are: KCl 76.04mg/l, MgSO 47H 2O 828.2mg/l, KH 2PO 4250.4mg/l, Ca (NO 3) 24H 2O 1001.3mg/l, CuSO 45H 2O 0.027mg/l, KI0.014mg/l, ZnSO 47H 2O0.027mg/l, H 3BO 30.309mg/l, Na 2MoO 42H 2O 0.012mg/l, MnCl 24H 2O0.198mg/l, CoCl 26H 2O 0.027mg/l, FeSO 47H 2O 27.8mg/l, Na 2-EDTA 37.3mg/l, tartaric acid ammonia 0.1151mg/l;
3) with step 2) expand numerous protonema that obtains and change over to and induce protonema to form on the gametophytic medium, to cultivate 40～60 days, condition of culture is: 25～30 ℃ of temperature, intensity of illumination 3000～4000lx, light application time 12～14 hours/day; Describedly induce protonema to form gametophytic culture medium prescription to be: improvement White medium+0～2.0mg/l 6-furfuryl group aminopurine+5～10g/l sucrose, wherein improve the White culture medium prescription and be: KNO 380mg/l, MgSO 47H 2O 720mg/l, NaH 2PO 4H 2O 16.5mg/l, KCl 65mg/l, Na 2SO 4200mg/l, Ca (NO 3) 24H 2O 300mg/l, CuSO 45H 2O 0.001mg/l, ZnSO 47H 2O 3.0mg/l, H 3BO 31.5mg/l, Na 2MoO 42H 2O 0.0001mg/l, MnSO 44H 2O7.0mg/l, FeNa-EDTA 4.588mg/l, inositol 100mg/l, nicotinic acid 0.3mg/l, thiamine hydrochloride 0.1mg/l, pyridoxine hydrochloride 0.1mg/l, glycine 3mg/l.
2. the gametophytic method of artificial expansion according to claim 1 numerous warm ground Da Ye moss, it is characterized in that, be the alcoholic solution of 70～75% volume fractions and the mercuric chloride solution of 0.1%～0.2% mass fraction to gametophyte surface disinfector for disinfecting in the described method.
3. the gametophytic method of artificial expansion according to claim 1 numerous warm ground Da Ye moss is characterized in that, the stem apex that cuts in the described method derives from the gametophyte that spire does not launch as yet, and length is 0.5～1.0cm.
4. the gametophytic method of artificial expansion according to claim 1 numerous warm ground Da Ye moss, it is characterized in that, described culture medium prescription of inducing gametophyte to produce protonema is: improvement Knop+2.0mg/l 2,4 dichlorophenoxyacetic acid+0.1mg/l 6-benzylaminopurine+5～10g/l sucrose.
5. the gametophytic method of artificial expansion according to claim 1 numerous warm ground Da Ye moss, it is characterized in that, the prescription that described protonema expands breeding culture medium is: improvement Knop medium+1.0mg/l 2,4 dichlorophenoxyacetic acid or 1.0mg/l 6-furfuryl group aminopurine+5～10g/l sucrose.
6. the gametophytic method of artificial expansion according to claim 1 numerous warm ground Da Ye moss, it is characterized in that, describedly induce protonema to form gametophytic culture medium prescription to be: improvement White medium+1.0mg/l6-furfuryl group aminopurine or additional any plant hormone+5～10g/l sucrose.
7. the gametophytic method of artificial expansion according to claim 1 numerous warm ground Da Ye moss is characterized in that the condition of culture in the described step 1) is: 23 ± 2 ℃ of temperature, and intensity of illumination 2500 ± 250lx, light application time 10 hours/day was cultivated 50 days.
8. the gametophytic method of the numerous expansion of artificial expansion according to claim 1 numerous warm ground Da Ye moss is characterized in that described step 2) in condition of culture be: 23 ± 2 ℃ of temperature, intensity of illumination 3000 ± 250lx, light application time 12 hours/day was cultivated 55 days.
9. the gametophytic method of artificial expansion according to claim 1 numerous warm ground Da Ye moss is characterized in that the condition of culture in the described step 3) is: 27 ± 2 ℃ of temperature, and intensity of illumination 3500 ± 250lx, light application time 14 hours/day was cultivated 60 days.
CN2008100346502A 2008-03-14 2008-03-14 Artificial enlarge method (of) Rhodobryum giganteum gametophyte Expired - Fee Related CN101248759B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN2008100346502A CN101248759B (en) 2008-03-14 2008-03-14 Artificial enlarge method (of) Rhodobryum giganteum gametophyte

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN2008100346502A CN101248759B (en) 2008-03-14 2008-03-14 Artificial enlarge method (of) Rhodobryum giganteum gametophyte

Publications (2)

Publication Number Publication Date
CN101248759A CN101248759A (en) 2008-08-27
CN101248759B true CN101248759B (en) 2011-05-25

Family

ID=39952505

Family Applications (1)

Application Number Title Priority Date Filing Date
CN2008100346502A Expired - Fee Related CN101248759B (en) 2008-03-14 2008-03-14 Artificial enlarge method (of) Rhodobryum giganteum gametophyte

Country Status (1)

Country Link
CN (1) CN101248759B (en)

Families Citing this family (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101606486B (en) * 2009-07-03 2011-05-25 上海师范大学 Method for fast propagating gametophyte of haplocladium microphyllum by tissue culture
CN103999772A (en) * 2014-05-20 2014-08-27 上海师范大学 In-vitro rapid propagation method of macromitrium cavaleriei card and ther
CN105981636B (en) * 2015-02-11 2019-03-29 上海市园林科学规划研究院 A kind of bog moss protonema rapid propagation method
CN105191802B (en) * 2015-10-27 2017-07-04 金陵科技学院 A kind of plant tissue capsule, preparation and application
CN113273494A (en) * 2021-06-17 2021-08-20 内蒙古大学 Culture medium for artificial propagation of wall moss and artificial propagation method
CN113207696A (en) * 2021-06-17 2021-08-06 内蒙古大学 Culture medium for artificial propagation of rhodobryum tassii and artificial propagation method
CN115152562B (en) * 2022-08-22 2023-09-08 云南三七科技有限公司 Planting method of Huixin grass

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1685109A (en) * 2002-10-02 2005-10-19 芝维纸业化工股份有限公司 Recyclable moisture proof packaging paper

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1685109A (en) * 2002-10-02 2005-10-19 芝维纸业化工股份有限公司 Recyclable moisture proof packaging paper

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
刘莹等.苔藓植物资源应用价值及展望.《安徽农业科学》.2007,第35卷(第28期),9000-9001. *
陈静文等.植物激素对小立碗藓愈伤组织诱导及分化影响.《上海师范大学(自然科学版)》.2006,第35卷(第4期),70-74. *
高永超等.苔藓植物的组织培养.《植物生理学通讯》.2002,第38卷(第6期),607-610. *

Also Published As

Publication number Publication date
CN101248759A (en) 2008-08-27

Similar Documents

Publication Publication Date Title
CN101248759B (en) Artificial enlarge method (of) Rhodobryum giganteum gametophyte
CN1319438C (en) Tissue culturing method for licorice adventitious root
CN102144554A (en) Method for producing lycoris by plant tissue culture
CN106577300B (en) The method for improving squalene content in Siraitia grosvenorii
CN108770691A (en) A method of induction camphor tree leaf blueberry tissue culture seedling leaf directly generates adventitious root
CN103483038B (en) Preparation and culture method of Cordyceps culture medium, and application thereof
CN102210265A (en) Method for cultivating adventitious roots of pseudo-ginseng
CN112544380A (en) Method for planting gastrodia elata by using substitute bacteria
CN1653889A (en) Method for preserving germplasm for Atractylis lancea tissue culture propagation
CN103548691B (en) The method of tea-tree tissue culture seedling culture of rootage
CN103141391A (en) Cultural method of American ginseng adventitious root tissue
US20220007604A1 (en) Methods of crossbreeding fungi organisms
CN104221862B (en) The method of medicinal Dioscorea camposita group training forming seedling through one step culture mass production
CN103548693B (en) Tissue culture rapid-multiplying method for bletilla striata protocorm-like bodies
CN103125397A (en) Culture medium for generating and growing medicinal woody plant callus and application thereof
CN102511393B (en) Method for establishing racomitrium japonicum gametophyte regeneration system
CN100420741C (en) Agrobacterium rhizogenes mediated aconitum coreanum and its obtaining method
CN106171996B (en) A kind of rapid propagation method of wild white birch
CN102550493A (en) Method for artificial infestation of Odontothrips loti on alfalfa
CN104774109A (en) Black fungus substitute material containing polygonum cuspidatum residues and preparation method of black fungus substitute material
CN1256870C (en) Notoginseng adventitious root tissue culture and its production method
CN103125383B (en) Method for setting up non-pollinated ovule regeneration system of rubber tree
CN106342623A (en) Ectotrophic mycorrhiza infecting method for high efficient symbiontic with raspberry
CN105432470A (en) Tissue culture method for dendrobium moniliforme
CN102301956B (en) Callus induction method taking paris polyphylla coleoptiles as explants

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
C14 Grant of patent or utility model
GR01 Patent grant
C17 Cessation of patent right
CF01 Termination of patent right due to non-payment of annual fee

Granted publication date: 20110525

Termination date: 20140314