CN109329048A - A kind of test tube seedling of roxburgh anoectochilus terminal bud germplasm grows store method slowly - Google Patents
A kind of test tube seedling of roxburgh anoectochilus terminal bud germplasm grows store method slowly Download PDFInfo
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
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Abstract
The present invention provides a kind of test tube seedlings of roxburgh anoectochilus terminal bud germplasm to grow store method slowly, method includes the following steps: the materials and disinfection of S1, explant；S2, inoculation and induction；S3, shoot proliferation；S4, the processing of test tube seedling low temperature stress；S5, test tube seedling grow preservation slowly；S6, test tube seedling rejuvenation；S7, rooting of vitro seedling；S8, transplanting.The microenvironment condition that the present invention is grown by changing germplasm materials; the speed of growth of test tube seedling is effectively delayed; extend the squamous subculture period; the preserving seed time is up to 3-4; and stabilization characteristics of genetics, germplasm survival rate is high, effect of rejuvenation is significant, is all of great significance to further protection roxburgh anoectochilus terminal bud resource and research, development and utilization roxburgh anoectochilus terminal bud.
Description
Technical field
The invention belongs to the medicinal plant biotechnologys in agricultural field, and in particular to a kind of test tube seedling of roxburgh anoectochilus terminal bud germplasm is slow
Grow store method.
Background technique
Roxburgh anoectochilus terminal bud (Anoectochilus roxburghii) i.e. Anoectochilus Roxburghii, alias gold silkworm, Shorthairy Antenoron, metal and stone pine,
Gold thread taiwan anetochilus herb, gold thread disappear to the marrow, are a kind of herbaceos perennials of orchid family Anoectochilus Blume；Its blade buckler-shaped, surface
It is dark brown, below kermesinus, flannelette shape is obvious, has golden netted vein entitled " gold thread ", leaf base sheath shape phimosis forms sheath
Section, matter is thick and solid tall and straight like lotus, thus roxburgh anoectochilus terminal bud of gaining the name.Roxburgh anoectochilus terminal bud happiness is shady and cool, moist, especially likes being grown in evergreen broad-leaved
The loose sough of the limes marginis of trees, cliff, soil property is typically distributed across 300 ~ 1200 meters of height above sea level of hilly ground.China exists more
The distribution of the provinces such as Fujian, Guangdong, Guangxi, Taiwan, Sichuan, Guizhou, Yunnan.Roxburgh anoectochilus terminal bud is rare medicinal plant, is had a variety of aobvious
The medical value and health-care efficacy of work.Roxburgh anoectochilus terminal bud is known as the laudatory titles such as " gold grass ", " god's medicine ", " black ginseng ", all herbal medicine, civil
Therapeutic domain is wider, has clearing heat and cooling blood, expelling wind and removing dampness, removing toxic substances, analgesic, Yin Yang balancing, strengthens the body resistance to consolidate the constitution, yin yang complementarity promotes the production of body fluid
Beauty treatment, the five internal organs, supports longevity macrobiosis and other effects at the regulation of qi and blood, cure mainly hemoptysis, bronchitis, ephritis, cystitis, diabetes, blood urine,
The illnesss such as rheumatic arthritis, acute, chronic hepatitis, myasthenia gravis.Recent studies indicate that roxburgh anoectochilus terminal bud contains volatile oil, more
Sugar, flavones, glycosides compound, steroidal compounds, organic acid, triterpenes, alkaloid, ucleosides, amino acid, microelement etc. its
His Multiple components, wherein polysaccharide, flavones, volatile oil are considered as the important component of roxburgh anoectochilus terminal bud drug effect, have enhancing immunity of organism
The effect of power, liver-protecting and cancer-resisting.There is good effect to the treatment and health care of disease just because of roxburgh anoectochilus terminal bud, market needs
The amount of asking is huge, and development prospect is extremely wide.
Under field conditions (factors), requirement of the roxburgh anoectochilus terminal bud to growing environment is extremely harsh, and it is complete, opposite to be often sporadicly distributed in vegetation
The hayashishita surface layer that humidity is high, humus is abundant, and roxburgh anoectochilus terminal bud seed does not have endosperm, and no germinating capacity is only total in fungi
It gives birth to, just promotes seed germination and growth, but germination percentage is very low, therefore wild resource update is very slow.In early days in the market
The source of goods of roxburgh anoectochilus terminal bud mostlys come from wild excavation, and long-term digging, which is adopted, makes its natural growing environment subject to severe risks of damage, and then leads
Wild gold lotus population quantity has been caused sharply to reduce.In recent years, roxburgh anoectochilus terminal bud artificial cultivation area expands rapidly, wild for making up
The deficiency of roxburgh anoectochilus terminal bud.But the provenance of artificial cultivation is mostly from wild resource, the roxburgh anoectochilus terminal bud of Different Provenances its plant height,
The chemical components such as Morphologic Characters and polysaccharide, flavones such as diameter, leaf length, plant fresh weight have differences, so that yield and inherence
Unstable quality has seriously affected the quality of medicinal material.
Excellent germ plasm resource is not only to improve the key of Chinese medicine quality and source or roxburgh anoectochilus terminal bud breed breeding and change
Good material base.Therefore carry out bud germ plasm resource evaluation study, and to the germ plasm resource of collection be subject to properly save and
It utilizes, is conducive to excavate and utilize the excellent inhereditary feature of roxburgh anoectochilus terminal bud, is an important process for pushing roxburgh anoectochilus terminal bud industry development.Mesh
Before, the tissue culture technique of roxburgh anoectochilus terminal bud reaches its maturity, and can carry out the Plantlet in vitro of germ plasm resource on this basis.Germplasm is in vitro
Saving is germplasm In Vitro conservation, and this method has many advantages: firstly, the growth of plant all in the environment of artificial control into
Row, this makes plant from the selection of disease pest and environmental weather conditions, to keep certain gene stability；Secondly, by from
Body culture can quantitatively form certain scale in a short time, can be used for breed improvement and breeding research at any time；Its
Three, in vitro culture material volume is small, and does not carry germ, thus is conducive to be transported between country and be exchanged.Mesh
The preservation of in Vitro Grown Shoot of preceding roxburgh anoectochilus terminal bud germplasm is not yet established, therefore carries out the depth to roxburgh anoectochilus terminal bud growth characteristics and tissue culture propagation
Enter research, exploration establishes long holding time, germplasm survival rate height, reliable and stable roxburgh anoectochilus terminal bud preserving seed method to roxburgh anoectochilus terminal bud production
The long term growth of industry has great importance.
Summary of the invention
Aiming at the problem that lacking effective bud germ plasm resource store method at present, the present invention provides a kind of roxburgh anoectochilus terminal bud kind
The test tube seedling of matter grows store method slowly, for saving the Wild ornamental resources of roxburgh anoectochilus terminal bud and the high-quality provenance of artificial cultivation,
Be conducive to the continuity of roxburgh anoectochilus terminal bud genetic diversity, also meet the needs that scientific development utilizes bud germ plasm resource.
The purpose of the present invention is what is solved by the following technical programs:
A kind of test tube seedling of roxburgh anoectochilus terminal bud germplasm grows store method slowly, which is characterized in that method includes the following steps:
The materials and disinfection of S1, explant
It selects that growing way is excellent, roxburgh anoectochilus terminal bud plant of no disease and pests harm, plant is cleaned with circulating water, removes root and blade, is retained
Stem section is as explant, and carry out disinfection processing in superclean bench；
S2, inoculation and induction
Explant after step S1 disinfection is cut into the stem-segment with node of 1-2cm, is inoculated in induced medium, in temperature 22-
24 DEG C, relative humidity 60-65%, intensity of illumination 1000-1400lx, induce Multiple Buds under conditions of light application time 6-8h/d, cultivate
Time is 25-30d；
S3, shoot proliferation
The step S2 Multiple Buds induced are cut into the fritter with 2-4 adventitious bud, are seeded on proliferated culture medium, every 21d
Subculture 1 time, the test tube seedling of subculture 2-3 times acquisition is as preserving seed material；Condition of culture control is 22-24 DEG C of temperature, relatively
Humidity 60-65%, intensity of illumination 1500-2000lx avoid direct projection, light application time 10-12h/d；
S4, the induction of test tube seedling alternating temperature salt stress
The step S3 test tube seedling obtained is placed in 3 DEG C of 25 DEG C of temperature, intensity of illumination 1500lx, illumination 14h and temperature, dark 10h
Then alternate culture 2d is 50mmol/LNaCl solution processing 1d with concentration, salt stress induces test tube seedling；
S5, test tube seedling grow preservation slowly
Treated test tube seedling is forwarded on preserving seed culture medium, one layer of sterile liquid stone is covered on culture medium
Wax, being subsequently placed under conditions of 10-15 DEG C of temperature, relative humidity 55-60%, intensity of illumination 400-600lx, daily illumination 12h makes
Test tube seedling is slowly grown, and every 12 months subcultures are primary, and subculture number is no more than 4 times；
S6, test tube seedling rejuvenation
After the roxburgh anoectochilus terminal bud preserving seed phase, the test tube seedling survived in above-mentioned steps, which is transferred in rejuvenation culture medium, makes its recovery
Growth, condition of culture control avoid direct projection for 23-27 DEG C of temperature, relative humidity 60-65%, intensity of illumination 1400-1800lx,
Daily illumination 12-14h, incubation time 17-23d；
S7, rooting of vitro seedling
When test tube seedling after rejuvenation grows to 4-5cm, seedling is cut into single plant and is transferred in root media, 23-27 DEG C of temperature,
Root induction under conditions of intensity of illumination 2500-3000lx, daily illumination 13-15h, incubation time 26-32d, it is seen that test tube
Miaoye color is dark green, and how sturdy root is；
S8, transplanting
Test tube seedling is transferred in hardening greenhouse together with tissue culture bottle, opens bottleneck adaptability hardening 7-10d；Then by test tube seedling from
It is carefully taken out in bottle, washes away the culture medium residual of root, root is impregnated in the liquor potassic permanganate that mass fraction is 0.01%
Then 10min is transplanted into the cultivation matrix of greenhouse, the environmental requirement of greenhouse is 23-28 DEG C of temperature, relative air humidity 80-
90%, scattering light irradiation, intensity of illumination 2600-3000lx, well-ventilated.
The method that explant is disinfected in the step S1 are as follows: first sterilize 30s, aseptic water washing 3-4 with 70% isopropanol
It is secondary, then 8min, aseptic water washing 4-6 times are sterilized with 0.1% mercuric chloride+3 drop polysorbas20；Finally use 500mg/L polyvinylpyrrolidone
6min is impregnated, filter paper sucks spare after excessive moisture.
The formula of induced medium is using MS as minimal medium in the step S2, add NAA 0.2mg/L,
TDZ0.4mg/L, lemon juice 53mL/L, lactoalbumin hydrolysate 5.8g/L, pH value are 5.6 ~ 6.0.
The formula of proliferated culture medium is using MS as minimal medium in the step S3, add 6-BA2.0mg/L,
NAA0.5mg/L, 2-(3,4- dichlorophenoxy) triethylamine 0.9mg/L, yeast extract 9g/L, pH value be 5.6 ~ 6.0.
The sterile liquid paraffin covered in the step S5 need to be higher by media surface 6-8mm.
The formula of preserving seed culture medium in the step S5 are as follows: KNO3940 ~ 1000mg/L, (NH 4 ) 2 SO 4 818~
830mg/L, CaCl2·2H2O 217 ~ 225mg/L, MgSO4·7H2O 180 ~ 190mg/L, KH2PO493 ~ 99mg/L,
MnSO4·4H2O 31 ~ 33mg/L, ZnSO4·7H2O 8.5 ~ 9.0mg/L, H3BO30.7 ~ 0.8mg/L of 5.9 ~ 6.3mg/L, KI,
CoCl2·6H2O 0.024 ~ 0.026mg/L, Na2MoO4·2H2O 33 ~ 35mg/L of 0.23 ~ 0.27mg/L, EDDHA-Fe, inositol
47 ~ 55mg/L, proline-4 ~ 6mg/L, 2 ~ 4mg/L of tryptophan, 2 ~ 4mg/L of methionine, 0.3 ~ 0.4mg/L of thiamine hydrochloride,
0.4 ~ 0.5mg/L of puridoxine hydrochloride, 0.3 ~ 0.4mg/L of vitamin B5,20 ~ 30 μm of ol/L of ancymidol, dimethyl succinic acid 4.8 ~
5.4mg/L, 6.0 ~ 6.4mg/L of tea polyphenols, acetylsalicylic acid 0.5 ~ 0.7mg/L, 5- 5.5 ~ 6.0mg/L of aza-cytidine,
35 ~ 40g/L of sucrose, 15 ~ 20mg/L of glycine betaine, 13 ~ 17mg/L of Quercetin, 5.2 ~ 5.6mg/L of hymexazol, peppermint leaching liquor 22 ~
28mL/L, 30 ~ 36mg/L of mashed carrot, plant gel 7 ~ 8g/L, pH value are 5.6 ~ 6.0.
The formula of rejuvenation culture medium in the step S6 are as follows: MS(is free of agar)+glutathione 11 ~ 15mg/L+ carboxymethyl
Sodium cellulosate 2.5 ~ 3.0g/L+ compound sodium nitrophenolate 1.2 ~ 1.6mg/L+ Titanium Citrate 0.4 ~ 0.8mg/L+N- acetyl -5- methoxy primary colours
1.1 ~ 1.3 μm of ol/L of amine+brassinosteroid 0.07 ~ 0.10mg/L+ plant gel 2 ~ 4g/L, pH value is 5.6 ~ 6.0.
The formula of root media in the step S7 is using 1/2MS as minimal medium, and wherein 7.5 g/L of agar, attached
Adding IBA0.5mg/L, microcystin 20mg/L, haydite 0.3g/L, pH value is 5.6 ~ 6.0.
The ingredient of cultivation matrix is garden mould by weight in the step S8: decomposed wood sawdust: sandy soil: bean cake powder: sugarcane
Slag=7:4:2:1:3.
In the preserving seed culture medium of the step S5, peppermint leaching liquor the preparation method comprises the following steps: fresh mint stem, leaf is taken to smash
It is broken, it is put into heat preservation extraction 2h in 60-70 DEG C of hot water for the material-water ratio of 1:4 in mass ratio, 200 mesh filtered through gauze of juice are removed
Solid residue, then sterile leaching liquor is made with sterilizing filter filtering in superclean bench.
Beneficial effects of the present invention have the following:
1) test tube seedling is placed in adverse circumstance appropriate and carries out stress-inducing, so that it is generated the response to environment-stress in vivo to enhance examination
The resistance of Guan Miao, and then improve the survival rate after test tube seedling long-term preservation.
2) key link of entire roxburgh anoectochilus terminal bud germplasm In Vitro conservation is improved, by reducing cultivation temperature, subtracting
Few intensity of illumination reduces the microenvironment that oxygen content changes preservation culture, optimizes nutrient media components, makes roxburgh anoectochilus terminal bud test tube seedling
Growth rate minimize limit, but do not lose vigor, to extend subculture interval time, reduce subculture number, make gold thread
The preserving seed period of lotus is up to 3-4.
3) test tube seedling overall survival rate remains to reach 59.5% after longest retention cycle, in life after rejuvenation culture
Root, transplanting aspect performance are good, are a kind of effective bud germ plasm resource store methods.
Specific embodiment
In order to be more clear the objectives, technical solutions, and advantages of the present invention, with reference to embodiments, the present invention is carried out
It is further described.It should be appreciated that specific embodiment described herein is only used to more fully understand technology of the invention
Scheme is not intended to limit the present invention.
Embodiment 1
2013-2017, the Fujian garden leaf roxburgh anoectochilus terminal bud introduced using jiangsu wuxi peace town roxburgh anoectochilus terminal bud plantation are pressed as test material
Preserving seed test is carried out according to the present invention, detailed step is as follows:
The materials and disinfection of S1, explant
It selects that growing way is excellent, roxburgh anoectochilus terminal bud plant of no disease and pests harm, plant is cleaned with circulating water, removes root and blade, is retained
Stem section first sterilizes 30s, aseptic water washing 3-4 times, then with 0.1% liter with 70% isopropanol in superclean bench as explant
Mercury+3 drips polysorbas20 and sterilizes 8min, aseptic water washing 4-6 times；Finally 6min, filter paper are impregnated with 500mg/L polyvinylpyrrolidone
It sucks spare after excessive moisture；
S2, inoculation and induction
Explant after step S1 disinfection is cut into the stem-segment with node of 1-2cm, is inoculated in induced medium (induced medium
Formula using MS as minimal medium, add NAA 0.2mg/L, TDZ0.4mg/L, lemon juice 53mL/L, lactoalbumin hydrolysate
5.8g/L, pH value are 5.8), under conditions of 23 DEG C of temperature, relative humidity 62%, intensity of illumination 1200lx, light application time 7h/d
Induce Multiple Buds, incubation time 25-30d；
S3, shoot proliferation
The step S2 Multiple Buds induced are cut into the fritter with 2-4 adventitious bud, are seeded on proliferated culture medium (proliferation training
The formula of base is supported using MS as minimal medium, adds 6-BA2.0mg/L, NAA0.5mg/L, 2-(3,4- dichlorophenoxy) three second
Amine 0.9mg/L, yeast extract 9g/L, pH value are 5.8) every 21d subculture 1 time, and the test tube seedling of subculture 2 times acquisitions is as kind of a quality guarantee
Deposit material；Condition of culture control is 23 DEG C of temperature, relative humidity 63% or so, intensity of illumination 1800lx, light application time 11h/d；
S4, the induction of test tube seedling alternating temperature salt stress
The step S3 test tube seedling obtained is placed in 3 DEG C of 25 DEG C of temperature, intensity of illumination 1500lx, illumination 14h and temperature, dark 10h
Then alternate culture 2d is 50mmol/LNaCl solution processing 1d with concentration, enhances the resistance of test tube seedling；
S5, test tube seedling grow preservation slowly
Treated test tube seedling is forwarded on preserving seed culture medium, one layer is covered in media surface and is higher by culture medium
The sterile liquid paraffin of surface 6-8mm is subsequently placed in 12 DEG C of temperature, relative humidity 58%, intensity of illumination 500lx, daily illumination
Grow test tube seedling slowly under conditions of 12h, every 12 months subcultures are primary, continue culture 7 months after subculture 3 times, count test tube
The overall survival rate of seedling；
The formula of preserving seed culture medium are as follows: KNO3970mg/L, (NH4)2SO4824mg/L, CaCl2·2H2O 221mg/L,
MgSO4·7H2O 185mg/L, KH2PO496mg/L, MnSO4·4H2O 32mg/L, ZnSO4·7H2O 8.7mg/L, H3BO3
6.1mg/L, KI 0.75mg/L, CoCl2·6H2O 0.025mg/L, Na2MoO4·2H2O 0.25mg/L, EDDHA-Fe
34mg/L, inositol 51mg/L, proline 5mg/L, tryptophan 3mg/L, methionine 3mg/L, thiamine hydrochloride 0.35mg/L, salt
Sour pyridoxol 0.45mg/L, vitamin B5 0.35mg/L, ancymidol 25 μm of ol/L, dimethyl succinic acid 5.1mg/L, tea polyphenols
6.2mg/L, acetylsalicylic acid 0.6mg/L, 5- aza-cytidine 5.8mg/L, sucrose 37.5g/L, glycine betaine 17.5mg/L,
Quercetin 15mg/L, hymexazol 5.4mg/L, peppermint leaching liquor 25mL/L, mashed carrot 33mg/L, plant gel 7.5g/L, pH
Value is 5.8；Wherein peppermint leaching liquor the preparation method comprises the following steps: fresh mint stem, leaf is taken to smash to pieces, be in mass ratio the material-water ratio of 1:4
It is put into heat preservation extraction 2h in 60-70 DEG C of hot water, 200 mesh filtered through gauze of juice are removed into solid residue, then in superclean bench
It is middle that sterile leaching liquor is made with sterilizing filter filtering；
S6, test tube seedling rejuvenation
The test tube seedling survived in above-mentioned steps is transferred in rejuvenation culture medium (the training after 7 months 3 years by roxburgh anoectochilus terminal bud kind quality guarantee
Support the formula of base are as follows: MS(is free of agar)+glutathione 13mg/L+ sodium carboxymethylcellulose 2.75g/L+ compound sodium nitrophenolate 1.4mg/
1.2 μm of ol/L of L+ Titanium Citrate 0.6mg/L+N- acetyl -5- methoxytryptamine+brassinosteroid 0.08mg/L+ plant gel
3g/L, pH value are 5.8) to make its restoration ecosystem, and condition of culture control is 25 DEG C of temperature, relative humidity 63%, and intensity of illumination is
1600lx, avoids direct projection, and daily illumination 13h, incubation time 17-23d observe test tube seedling growing way；
S7, rooting of vitro seedling
When test tube seedling after rejuvenation grows to 4-5cm, seedling is cut into single plant and is transferred in root media that (root media is matched
Side is using 1/2MS as minimal medium, and wherein 3.5 g/L of agar, adds IBA0.5mg/L, microcystin 20mg/L, haydite
0.3g/L, pH value are 5.6 ~ 6.0), root induction under conditions of 25 DEG C of temperature, intensity of illumination 2800lx, daily illumination 14h,
Incubation time is 26-32d, counts rooting of vitro seedling rate, number of averagely taking root；
S8, transplanting
Test tube seedling is transferred in hardening greenhouse together with tissue culture bottle, opens bottleneck adaptability hardening 7-10d；Then by test tube seedling from
It is carefully taken out in bottle, washes away the culture medium residual of root, root is impregnated in the liquor potassic permanganate that mass fraction is 0.01%
Then 10min is transplanted into the cultivation matrix of greenhouse, the ingredient of cultivation matrix is garden mould by weight: decomposed wood sawdust: sandy
Soil: bean cake powder: bagasse=7:4:2:1:3, the environmental requirement of greenhouse are 23-28 DEG C of temperature, relative air humidity 80-90%, scattering
Light irradiation, intensity of illumination 2600-3000lx, well-ventilated；Transplanting survival rate is counted after transplanting 30d.
Embodiment 2
In order to prove influence of the stress-inducing method to preserving seed effect, the present embodiment eliminates step S3 in embodiment 1,
Remaining step and embodiment 1 are consistent, repeat no more.
Embodiment 3
In order to prove the influence of preserving seed culture medium of the invention to preserving seed effect, the present embodiment is by 1 step of embodiment
Preserving seed culture medium replaces with MS culture medium in S5, remaining step and embodiment 1 are consistent, repeats no more.
Embodiment 4
In order to prove the influence of germplasm rejuvenation culture medium of the invention to preserving seed effect, the present embodiment is by 1 step of embodiment
Rejuvenation culture medium replaces with MS culture medium in S6, remaining step and embodiment 1 are consistent, repeats no more.
As a result it counts
1, after preserving seed 7 months 3 years, statistical data every in embodiment 1-4 is summarized as follows table 1.
The test tube seedling survival rate of the data of comparing embodiment 1 and embodiment 2, discovery embodiment 1 is apparently higher than embodiment 2,
Illustrate that alternating temperature salt stress induction of the invention is effective, depositing for long-term preservation can be improved by enhancing test tube seedling resistance
Motility rate；The all data index of the data of comparing embodiment 1 and embodiment 3 and 4, discovery embodiment 1 is better than other embodiments,
Illustrate that preserving seed culture medium and germplasm rejuvenation culture medium of the invention are all the optimization formulas by further investigation, to roxburgh anoectochilus terminal bud
Preserving seed and restoration ecosystem all there are best effects.
The preferred embodiment of the present invention has been described above in detail, still, during present invention is not limited to the embodiments described above
Detail within the scope of the technical concept of the present invention can be with various simple variants of the technical solution of the present invention are made, this
A little simple variants all belong to the scope of protection of the present invention.
Claims (10)
1. a kind of test tube seedling of roxburgh anoectochilus terminal bud germplasm grows store method slowly, which is characterized in that method includes the following steps:
The materials and disinfection of S1, explant
It selects that growing way is excellent, roxburgh anoectochilus terminal bud plant of no disease and pests harm, plant is cleaned with circulating water, removes root and blade, is retained
Stem section is as explant, and carry out disinfection processing in superclean bench；
S2, inoculation and induction
Explant after step S1 disinfection is cut into the stem-segment with node of 1-2cm, is inoculated in induced medium, in temperature 22-
24 DEG C, relative humidity 60-65%, intensity of illumination 1000-1400lx, induce Multiple Buds under conditions of light application time 6-8h/d, cultivate
Time is 25-30d；
S3, shoot proliferation
The step S2 Multiple Buds induced are cut into the fritter with 2-4 adventitious bud, are seeded on proliferated culture medium, every 21d
Subculture 1 time, the test tube seedling of subculture 2-3 times acquisition is as preserving seed material；Condition of culture control is 22-24 DEG C of temperature, relatively
Humidity 60-65%, intensity of illumination 1500-2000lx avoid direct projection, light application time 10-12h/d；
S4, the induction of test tube seedling alternating temperature salt stress
The step S3 test tube seedling obtained is placed in 3 DEG C of 25 DEG C of temperature, intensity of illumination 1500lx, illumination 14h and temperature, dark 10h
Then alternate culture 2d is 50mmol/LNaCl solution processing 1d with concentration, salt stress induces test tube seedling；
S5, test tube seedling grow preservation slowly
Treated test tube seedling is forwarded on preserving seed culture medium, one layer of sterile liquid stone is covered on culture medium
Wax, being subsequently placed under conditions of 10-15 DEG C of temperature, relative humidity 55-60%, intensity of illumination 400-600lx, daily illumination 12h makes
Test tube seedling is slowly grown, and every 12 months subcultures are primary, and subculture number is no more than 4 times；
S6, test tube seedling rejuvenation
After the roxburgh anoectochilus terminal bud preserving seed phase, the test tube seedling survived in above-mentioned steps, which is transferred in rejuvenation culture medium, makes its recovery
Growth, condition of culture control avoid direct projection for 23-27 DEG C of temperature, relative humidity 60-65%, intensity of illumination 1400-1800lx,
Daily illumination 12-14h, incubation time 17-23d；
S7, rooting of vitro seedling
When test tube seedling after rejuvenation grows to 4-5cm, seedling is cut into single plant and is transferred in root media, 23-27 DEG C of temperature,
Root induction under conditions of intensity of illumination 2500-3000lx, daily illumination 13-15h, incubation time 26-32d, it is seen that test tube
Miaoye color is dark green, and how sturdy root is；
S8, transplanting
Test tube seedling is transferred in hardening greenhouse together with tissue culture bottle, opens bottleneck adaptability hardening 7-10d；Then by test tube seedling from
It is carefully taken out in bottle, washes away the culture medium residual of root, root is impregnated in the liquor potassic permanganate that mass fraction is 0.01%
Then 10min is transplanted into the cultivation matrix of greenhouse, the environmental requirement of greenhouse is 23-28 DEG C of temperature, relative air humidity 80-
90%, scattering light irradiation, intensity of illumination 2600-3000lx, well-ventilated.
2. a kind of test tube seedling of roxburgh anoectochilus terminal bud germplasm according to claim 1 grows store method slowly, which is characterized in that described
The method that explant is disinfected in step S1 are as follows: first with 70% isopropanol disinfection 30s, aseptic water washing 3-4 times, then with 0.1%
Mercuric chloride+3 drips polysorbas20 and sterilizes 8min, aseptic water washing 4-6 times；Finally 6min, filter are impregnated with 500mg/L polyvinylpyrrolidone
Paper sucks spare after excessive moisture.
3. a kind of test tube seedling of roxburgh anoectochilus terminal bud germplasm according to claim 1 grows store method slowly, which is characterized in that described
The formula of induced medium adds NAA 0.2mg/L, TDZ0.4mg/L, lemon juice using MS as minimal medium in step S2
53mL/L, lactoalbumin hydrolysate 5.8g/L, pH value are 5.6 ~ 6.0.
4. a kind of test tube seedling of roxburgh anoectochilus terminal bud germplasm according to claim 1 grows store method slowly, which is characterized in that described
The formula of proliferated culture medium adds 6-BA2.0mg/L, NAA0.5mg/L, 2-(3,4- bis- using MS as minimal medium in step S3
Chlorophenoxy) triethylamine 0.9mg/L, yeast extract 9g/L, pH value be 5.6 ~ 6.0.
5. a kind of test tube seedling of roxburgh anoectochilus terminal bud germplasm according to claim 1 grows store method slowly, which is characterized in that described
The sterile liquid paraffin covered in step S5 need to be higher by media surface 6-8mm.
6. a kind of test tube seedling of roxburgh anoectochilus terminal bud germplasm according to claim 1 grows store method slowly, which is characterized in that described
The formula of preserving seed culture medium in step S5 are as follows: KNO3940 ~ 1000mg/L, (NH 4 ) 2 SO 4818 ~ 830mg/L,
CaCl2·2H2O 217 ~ 225mg/L, MgSO4·7H2O 180 ~ 190mg/L, KH2PO493 ~ 99mg/L, MnSO4·4H2O 31
~ 33mg/L, ZnSO4·7H2O 8.5 ~ 9.0mg/L, H3BO35.9 ~ 6.3mg/L, KI 0.7 ~ 0.8mg/L, CoCl2·6H2O
0.024 ~ 0.026mg/L, Na2MoO4·2H2O 0.23 ~ 0.27mg/L, EDDHA-Fe 33 ~ 35mg/L, 47 ~ 55mg/L of inositol,
Proline-4 ~ 6mg/L, 2 ~ 4mg/L of tryptophan, 2 ~ 4mg/L of methionine, 0.3 ~ 0.4mg/L of thiamine hydrochloride, puridoxine hydrochloride
0.4 ~ 0.5mg/L, 0.3 ~ 0.4mg/L of vitamin B5, ancymidol 20 ~ 30 μm of ol/L, 4.8 ~ 5.4mg/L of dimethyl succinic acid, tea
6.0 ~ 6.4mg/L of polyphenol, acetylsalicylic acid 0.5 ~ 0.7mg/L, 5- 5.5 ~ 6.0mg/L of aza-cytidine, 35 ~ 40g/ of sucrose
L, 15 ~ 20mg/L of glycine betaine, 13 ~ 17mg/L of Quercetin, 5.2 ~ 5.6mg/L of hymexazol, peppermint 22 ~ 28mL/L of leaching liquor, carrot
30 ~ 36mg/L of mud, plant gel 7 ~ 8g/L, pH value are 5.6 ~ 6.0.
7. a kind of test tube seedling of roxburgh anoectochilus terminal bud germplasm according to claim 1 grows store method slowly, which is characterized in that described
The formula of rejuvenation culture medium in step S6 are as follows: MS(is free of agar)+11 ~ 15mg/L+ of glutathione sodium carboxymethylcellulose 2.5 ~
1.1 ~ 1.3 μ of 3.0g/L+ compound sodium nitrophenolate 1.2 ~ 1.6mg/L+ Titanium Citrate 0.4 ~ 0.8mg/L+N- acetyl -5- methoxytryptamine
Mol/L+brassinosteroid 0.07 ~ 0.10mg/L+ plant gel 2 ~ 4g/L, pH value is 5.6 ~ 6.0.
8. a kind of test tube seedling of roxburgh anoectochilus terminal bud germplasm according to claim 1 grows store method slowly, which is characterized in that described
The formula of root media in step S7 is using 1/2MS as minimal medium, and wherein 7.5 g/L of agar, adds IBA0.5mg/
L, microcystin 20mg/L, haydite 0.3g/L, pH value are 5.6 ~ 6.0.
9. a kind of test tube seedling of roxburgh anoectochilus terminal bud germplasm according to claim 1 grows store method slowly, which is characterized in that described
The ingredient of cultivation matrix is garden mould by weight in step S8: decomposed wood sawdust: sandy soil: bean cake powder: bagasse=7:4:2:1:3.
10. a kind of test tube seedling of roxburgh anoectochilus terminal bud germplasm according to claim 5 grows store method slowly, which is characterized in that institute
State in the preserving seed culture medium of step S5, peppermint leaching liquor the preparation method comprises the following steps: taking fresh mint stem, leaf to smash to pieces, by quality
It is put into heat preservation extraction 2h in 60-70 DEG C of hot water than the material-water ratio for 1:4,200 mesh filtered through gauze of juice are removed into solid residue,
Sterile leaching liquor is made with sterilizing filter filtering in superclean bench again.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110089407A (en) * | 2019-04-04 | 2019-08-06 | 福建省农业科学院作物研究所 | A kind of bagging plantation store method of bud germ plasm resource |
| CN110089427A (en) * | 2019-04-09 | 2019-08-06 | 福建省农业科学院作物研究所 | A kind of in-vitro conservation method of bud germ plasm resource |
| CN112493134A (en) * | 2020-12-17 | 2021-03-16 | 刘泽洋 | In-vitro open culture method for plants and in-vitro open special culture medium |
| CN112493133A (en) * | 2020-12-17 | 2021-03-16 | 刘泽洋 | Plant open tissue culture method and special culture medium |
| CN113100059A (en) * | 2021-04-19 | 2021-07-13 | 福建农林大学 | Long-term in-vitro preservation method for anoectochilus formosanus |
-
2018
- 2018-09-25 CN CN201811117082.2A patent/CN109329048A/en not_active Withdrawn
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110089407A (en) * | 2019-04-04 | 2019-08-06 | 福建省农业科学院作物研究所 | A kind of bagging plantation store method of bud germ plasm resource |
| CN110089407B (en) * | 2019-04-04 | 2021-09-07 | 福建省农业科学院作物研究所 | Bagging planting and storing method for anoectochilus roxburghii germplasm resources |
| CN110089427A (en) * | 2019-04-09 | 2019-08-06 | 福建省农业科学院作物研究所 | A kind of in-vitro conservation method of bud germ plasm resource |
| CN110089427B (en) * | 2019-04-09 | 2022-06-10 | 福建省农业科学院作物研究所 | In-vitro preservation method of anoectochilus roxburghii germplasm resources |
| CN112493134A (en) * | 2020-12-17 | 2021-03-16 | 刘泽洋 | In-vitro open culture method for plants and in-vitro open special culture medium |
| CN112493133A (en) * | 2020-12-17 | 2021-03-16 | 刘泽洋 | Plant open tissue culture method and special culture medium |
| CN112493133B (en) * | 2020-12-17 | 2022-03-01 | 青岛泽欣农业科技有限公司 | Plant open tissue culture method and special culture medium |
| CN112493134B (en) * | 2020-12-17 | 2022-05-20 | 内蒙古宏源农牧业科技股份有限公司 | In-vitro open culture method for plants and special in-vitro open culture medium |
| CN113100059A (en) * | 2021-04-19 | 2021-07-13 | 福建农林大学 | Long-term in-vitro preservation method for anoectochilus formosanus |
| CN113100059B (en) * | 2021-04-19 | 2022-08-16 | 福建农林大学 | Long-term in-vitro preservation method for anoectochilus formosanus |
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