CN102265788B - Method for increasing proliferation multiple of dendrobium candidum protocorms - Google Patents
Method for increasing proliferation multiple of dendrobium candidum protocorms Download PDFInfo
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- CN102265788B CN102265788B CN2011102004982A CN201110200498A CN102265788B CN 102265788 B CN102265788 B CN 102265788B CN 2011102004982 A CN2011102004982 A CN 2011102004982A CN 201110200498 A CN201110200498 A CN 201110200498A CN 102265788 B CN102265788 B CN 102265788B
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Abstract
The invention relates to a method for increasing the proliferation multiple of dendrobium candidum protocorms, belonging to the technical field of biological engineering. The method comprises the following steps of: preparation of a culture medium: preparing the culture medium in a culture vessel, and sterilizing the culture medium at high temperature to obtain the culture vessel containing the culture medium; inoculation: inoculating 0.5 grams of the dendrobium candidum protocorms inside a female bottle or 0.5 grams of protocorms obtained after the dendrobium candidum protocorms are subcultured into the culture vessel with the culture medium to obtain a culture medium inoculation culture vessel; magnetic treatment: placing the culture medium inoculation culture vessel into a magnetic field for the magnetic treatment, controlling magnetic treatment time and magnetic induction density to obtain the culture medium inoculation culture vessel subjected to the magnetic treatment; propagation culture: placing the culture medium inoculation culture vessel subjected to the magnetic treatment into a culture room for the propagation culture, and controlling the culturing temperature, the illumination intensity and the illumination time of the culture room to obtain the dendrobium candidum protocorms with 3-9 weight proliferation multiples. The method disclosed by the invention has the advantages that the proliferation multiples reaches 3-9 by utilizing magnetic field treatment.
Description
Technical field
The invention belongs to technical field of bioengineering, be specifically related to a kind of method that improves dendrobium candidum protocorm propagation multiple
Background technology
The morphological feature of dendrobium candidum: dendrobium candidum (
Dendrobium Candidum Wall.ex Lindl) being commonly called as Tiepi Fengdou, the orchid family is grown nonparasitically upon another plant, and stem is upright, it is cylindrical to grow thickly, green or steel gray; High several centimetres are not waited to tens centimetres, more piece, and internode expands, the obvious gloss of pitchy tool.Leaf minority, alternate be in stem top, stockless, and the oval shape of blade drapes over one's shoulders the pin type; Leaf sheath is membranous, hugs internode, canescence.Raceme is spent 2～5, pistac or faint yellow.Fruit is a capsule, and long ovum shape is dark green to dark green; Seed is little and many, and planting skin is the parenchyma cell of layer of transparent, and the ring grain of thickening is arranged, and plants skin and contains a large amount of air; Embryonic development is incomplete or immature in kind, no endosperm.Root is grown nonparasitically upon another plant on rock, and is oblate for unramified aerial root, white, and green or canescence, grey robe look, about thick 1 mm, length and plant height are general.
The growth characteristics of
dendrobium candidum: wild dendrobium candidum grows between height above sea level 900～1500 m more, in the evergreen broad-leaved forest of 12～18 ℃ of average temperatures of the whole year, relative moisture 60%～75%, light transmittance about 60%, 20～28 ℃ of season of growth temperature, 9～12 ℃ of winter temperatures, frostless foggy, annual rainfall 500～1000 mm.Dendrobium candidum nature ripening rate is very low, is main with cross pollination, and its self-pollination success rate is extremely low, only about 30%, and the cross pollination rate reaches 100% success.The survival rate of dendrobium candidum wild seed is very low.
The medical value of
dendrobium candidum: dendrobium candidum is as China's traditional Chinese medicine; Be divided into iron sheet, yellow grass, Jin Chai, Huoshan, etc. the dozens of kind; Wherein dendrobium candidum is the most precious, occupy first of " Chinese nine immortals grass ", and be the superfine product in the stem of noble dendrobium.Archaism cloud: " there is genseng in north, the Nan Youfeng bucket ", the laudatory title that " the celestial grass of help ", " the celestial grass of China ", " gold in the medicine " are also arranged among the people.Main product is in Yunnan Province of China, Guizhou, Zhejiang, Anhui and other places, and ten minutes is tender and lovely, and chance high light, heavy rain or snow freeze promptly can be dead, and excess moisture also can make its butt rot, even whole strain is dead.
cause long-term immoderate harvesting because dendrobium candidum has very high medical value, cause wild resource to be on the verge of exhaustion.And its natural propagation power is extremely low again, and conventional breeding is also difficult, and wild dendrobium candidum has been on the verge of disappearance.Country in 1987 has classified dendrobium candidum as and has laid special stress on protecting wild rare medicinal material kind, and country's first " Chinese rare and endangered protective plant register " is also listed it wherein in.In order to protect the wild resource of dendrobium candidum better, become wild and plant into family, should greatly develop the large-scale artificial cultivation, ensure the dendrobium candidum Sustainable utilization of resources conscientiously.From the seventies in 20th century, both at home and abroad appropriate authority has just begun the tame R&D work of dendrobium candidum, is especially carrying out big quantity research aspect the tissue culture of dendrobium candidum and quick propagating technology, the artificial cultivation technique.In dendrobium candidum tissue culture and quick propagating technology, the propagation multiple of protocorm is a restriction seedling development key.
Chinese invention patent Granted publication CN1275513C has recommended Ironbark dendrobe sprout; This patent scheme can embody 3 technique effects of the 4th page of conclusion of specification, but does not have high cultivation effect (propagation 1-2 doubly).Again; CN101180949B and CN200710066480.1 have all introduced a kind of seedling-cultivating method of dendrobium candidum stem apex quick breeding by group culture; The positive effect of this two patents scheme is: can shorten the cultivation cycle of dendrobium candidum seedling, make the cultivation cycle from the annulus stem to the seedling of taking root foreshorten to 5-6 month; Stabilization characteristics of genetics.But CN101180949B and CN200710066480.1 have only described the dendrobium candidum a kind of common seedling-cultivating method of breeding fast, except with the medium, do not disclose the concrete measure of dendrobium candidum weightening finish.And the fast reproducing method for high quality seedling of dendrobium officinale that CN101258835B discloses has equally only been described dendrobium candidum a kind of commonsense method of breeding fast, does not instruct the concrete measure of dendrobium candidum weightening finish.
Such as industry the reason of knowledge; The key of dendrobium candidum tissue culture and breeding depends on the propagation multiple of protocorm; Do not enlighten and in being not limited to, all provide corresponding techniques by above-mentioned disclosed patent documentation; For this reason, the applicant has made good try, and the technical scheme that will introduce below produces under this background
Summary of the invention
task of the present invention is to provide the method for the raising dendrobium candidum protocorm propagation multiple that ensures excellent cultivation effect in a kind of tissue culture and the reproductive process.
Task of the present invention is accomplished like this, and a kind of method that improves dendrobium candidum protocorm propagation multiple may further comprise the steps:
A) preparing culture medium, preparing culture medium in culture vessel, and, obtain filling the culture vessel of medium with this medium high-temperature sterilization;
B) inoculation perhaps will be inoculated in female bottle dendrobium candidum protocorm 0.5g in the culture vessel that fills medium by the protocorm 0.5g that obtains behind the dendrobium candidum protocorm successive transfer culture, obtain the culture medium inoculated culture vessel;
C) magnetic treatment places the magnetic field magnetic treatment with the culture medium inoculated culture vessel, controls magnetic treatment time and magnetic induction intensity, obtains the culture medium inoculated culture vessel of magnetic treatment;
D) enrichment culture; The culture medium inoculated culture vessel of magnetic treatment is placed culturing room's enrichment culture; And cultivation temperature, illuminance and the light application time of control culturing room, obtaining weight propagation multiple is 3-9 dendrobium candidum protocorm doubly.
are in a concrete embodiment of the present invention; Steps A) medium described in is: 1/2 MS+NAA (methyl) 0.2-0.4mg/L+6-BA (6-benzyladenine) 0.8-1 mg/L+KT (kinetin) 0.8-1 mg/L+ sucrose 20-40g/L+ agar 7-9g/L+ powder activated carbon 1-3g/L, pH5.6-5.8; Described high-temperature sterilization is said medium to be placed in the high-pressure sterilizing pot temperature is risen to 115-125 ℃, and pressure is 0.07-0.135MPa, and is cooled to room temperature after keeping 20-25min.
In another concrete embodiment of the present invention; Step B) the female bottle dendrobium candidum protocorm described in is to get the capsule that dendrobium candidum does not have cracking; Using mass percent concentration earlier is 75% ethanol wipe surfaces, and putting into mass percent concentration again is the mercury chloride (HgCl of 0.1%-0.2%
2
) soak 7-12min in the solution, clean with aqua sterilisa then, then cut capsule open, get its embryo and embryo is seeded on the MS medium of improvement and cultivate until the protocorm that grows; The MS medium of improvement is meant in the MS medium NH
4
NO
3
Use reduces by half; Described aqua sterilisa is meant distilled water or ordinary tap water placed and is warming up to 115-125 ℃ in the high-pressure sterilizing pot that pressure is 0.07-0.135MPa, and keeps 20-25min, again through being cooled to the resulting water of room temperature; The number of times of described cleaning is 3 times, 4 times or 5 times.
are in another concrete embodiment of the present invention; Step C) the control magnetic treatment time described in is that the magnetic treatment time is controlled to be 60-900min, and described control magnetic induction intensity is that magnetic induction intensity is controlled to be 6-25mT (milli tesla).
in another concrete embodiment of the present invention, step D) described in culturing room in time of enrichment culture be 30-60 days; The described cultivation temperature of controlling culturing room is that temperature is controlled to be 20-25 ℃, and described control illuminance is that illuminance is controlled to be 1000-2000LUX, and described control light application time is that light application time is controlled to be 12h/ days.
A kind of method that improves dendrobium candidum protocorm propagation multiple may further comprise the steps:
A) preparing culture medium, preparing culture medium in culture vessel, and, obtain filling the culture vessel of the medium that contains tri-iron tetroxide with this medium high-temperature sterilization;
B) inoculation; Female bottle dendrobium candidum protocorm 0.5g perhaps will be inoculated in the culture vessel that fills the medium that contains tri-iron tetroxide by the protocorm 0.5g that obtains behind the dendrobium candidum protocorm successive transfer culture, obtain containing the culture medium inoculated culture vessel of tri-iron tetroxide;
C) magnetic treatment, the culture medium inoculated culture vessel that will contain tri-iron tetroxide places the magnetic field magnetic treatment, controls magnetic treatment time and magnetic induction intensity, obtains the culture medium inoculated culture vessel that contains tri-iron tetroxide of magnetic treatment;
D) enrichment culture; The culture medium inoculated culture vessel that contains tri-iron tetroxide of magnetic treatment is placed culturing room's enrichment culture; And cultivation temperature, illuminance and the light application time of control culturing room, obtaining weight propagation multiple is 3-8.5 dendrobium candidum protocorm doubly.
Also have among the concrete embodiment steps A of the present invention) described in medium be: 1/2 MS+NAA (methyl) 0.2-0.4mg/L+6-BA (6-benzyladenine) 0.8-1 mg/L+KT (kinetin) 0.8-1 mg/L+ tri-iron tetroxide (Fe
3
O
4
) 2-6g/L+sucrose 20-40g/L+ agar 7-9g/L, pH5.6-5.8; Described high-temperature sterilization is said medium to be placed in the high-pressure sterilizing pot temperature is risen to 115-125 ℃, and pressure is 0.07-0.135MPa, and is cooled to room temperature after keeping 20-25min.
more of the present invention and among concrete embodiment; Step B) the female bottle dendrobium candidum protocorm described in is to get the capsule that dendrobium candidum does not have cracking; Using mass percent concentration earlier is 75% ethanol wipe surfaces, and putting into mass percent concentration again is the mercury chloride (HgCl of 0.1%-0.2%
2
) soak 7-12min in the solution, clean with aqua sterilisa then, then cut capsule open, get its embryo and embryo is seeded on the MS medium of improvement and cultivate until the protocorm that grows; The MS medium of improvement is meant in the MS medium NH
4
NO
3
Use reduces by half; Described aqua sterilisa is meant distilled water or ordinary tap water placed and is warming up to 115-125 ℃ in the high-pressure sterilizing pot that pressure is 0.07-0.135MPa, and keeps 20-25min, again through being cooled to the resulting water of room temperature; The number of times of described cleaning is 3 times, 4 times or 5 times.
are in of the present invention and then concrete embodiment; Step C) the control magnetic treatment time described in is that the magnetic treatment time is controlled to be 60-900min, and described control magnetic induction intensity is that magnetic induction intensity is controlled to be 6-25mT (milli tesla).
of the present invention again more and among concrete embodiment, step D) described in culturing room in time of enrichment culture be 30-50 days; The described cultivation temperature of controlling culturing room is that temperature is controlled to be 20-25 ℃, and described control illuminance is that illuminance is controlled to be 1000-2000LUX, and described control light application time is that light application time is controlled to be 12h/ days.
technical scheme provided by the invention has remedied the defective of the dendrobium candidum protocorm cultivation effect difference in the prior art, utilizes magnetic field treated and makes the propagation multiple reach 3-9 doubly.
Embodiment
Below embodiment in the MS that mentions be a kind of general basal medium, its prescription by macroelement, trace element, molysite and organic substance totally four types of materials form, the concrete consumption of each material is (mg/L of unit) as follows:
One, macroelement:
Ammonium nitrate (NH
4
NO
3
) 1650
Potassium nitrate (KNO
3
) 1900
Calcium chloride (CaCl
2
2H
2
O) 440
Magnesium sulfate (MgSO
4
7H
2
O) 370
Biphosphate first (KH
2
PO
4
) 170
Two, trace element:
KI (KI) 0.83
Boric acid (H
3
BO
3
) 6.2
Manganese sulphate (MnSO
4
4H
2
O) 22.3
Zinc sulphate (ZnSO
4
7H
2
O) 8.6
Sodium molybdate (Na
2
MoO
4
2H
2
O) 0.25
Copper sulphate (CuSO
4
5 H
2
O) 0.025
Cobalt chloride (CoCl
2
6 H
2
O) 0.025
Three, molysite:
Ferrous sulfate (FeSO
4
7 H
2
O) 27.8
Disodium ethylene diamine tetraacetate (Na
2
EDTA) 37.3
Four, organic substance:
Inositol 1
Nicotinic acid 0.5
Puridoxine hydrochloride (vitamin B6) 0.5
Thiamine hydrochloride (vitamin B1) 0.1
Glycine 2.0
1/2MS is meant that the consumption of ammonium nitrate in the aforesaid MS medium, potassium nitrate, calcium chloride, magnesium sulfate, these five kinds of materials of biphosphate first reduces by half, and promptly is respectively 825,950,220,185,85 mg/L; The MS medium of improvement is to the NH in the aforesaid MS medium
4
NO
3
Usage amount to reduce by half be 825mg/L.
Explain
: the MS medium is merely basal medium, also will add during use after the compositions such as 6-BA, sucrose, agar, just arrives 5.6-5.8 to pH regulator.
Embodiment 1:
A) preparing culture medium; Preparing culture medium in blake bottle, medium is: 1/2 MS+ NAA (naa) 0.2mg/L+ 6-BA (6-benzyladenine) 1mg/L+KT (kinetin) 1mg/L+ sucrose, 20 g/L+ agar 8g/L+ powder activated carbon 2g/L, the pH value is 5.6; With this medium high-temperature sterilization; The concrete grammar of high-temperature sterilization is: medium is placed on is warming up to 125 ℃ in the high-pressure sterilizing pot, pressure is 0.135MPa, and under this temperature and pressure, keeps 20min; To be cooled to room temperature, obtain filling the blake bottle of medium;
B) inoculation; Female bottle dendrobium candidum protocorm 0.5g is inoculated in by steps A) culture vessel that fills medium that obtains is in the blake bottle; Obtain the culture medium inoculated blake bottle; Female bottle dendrobium candidum protocorm described in this step is meant: get the capsule that dendrobium candidum does not have cracking, using mass percent concentration earlier is 75% ethanol wipe surfaces, and putting into mass percent concentration again is the mercury chloride (HgCl of 0.1%-0.2%
2
) soak 7-12min in the solution; (aqua sterilisa is meant distilled water or ordinary tap water be placed in the high-pressure sterilizing pot temperature is risen to 115-125 ℃ to use aqua sterilisa then; Corresponding therewith pressure is 0.07-0.135MPa), and be cooled to room temperature cleaning 3 times, 4 times or 5 times after keeping 20-25min, cut capsule open; Get its embryo and embryo is seeded on the MS medium of improvement the protocorm that cultivation grew in 1-2 month;
C) magnetic treatment will be by step B) the culture medium inoculated blake bottle that obtains places the magnetic field magnetic treatment, and the magnetic treatment time is 900min, and magnetic induction intensity is 12mT (milli tesla), obtains the culture medium inoculated blake bottle of magnetic treatment;
D) enrichment culture; Will be by step C) the culture medium inoculated blake bottle of the magnetic treatment that obtains places culturing room's enrichment culture; Culturing room's temperature is 25 ℃ of daytimes, and at 20 ℃ of nights, illuminance is 1100LUX; Light application time is 12h/ days, obtains weight propagation multiple and be 3.1 times dendrobium candidum protocorm.
Embodiment 2:
A) preparing culture medium; Preparing culture medium in blake bottle, medium is: 1/2 MS+ NAA (naa) 0.3mg/L+ 6-BA (6-benzyladenine) 1mg/L+KT (kinetin) 0.8mg/L+ sucrose, 30 g/L+ agar 9g/L+ powder activated carbon 1g/L, the pH value is 5.8; With this medium high-temperature sterilization; The concrete grammar of high-temperature sterilization is: medium is placed on is warming up to 115 ℃ in the high-pressure sterilizing pot, pressure is 0.07MPa, and under this temperature and pressure, keeps 25min; To be cooled to room temperature, obtain filling the blake bottle of medium;
B) inoculation; Female bottle dendrobium candidum protocorm 0.5g is inoculated in by steps A) culture vessel that fills medium that obtains is in the blake bottle; Obtain the culture medium inoculated blake bottle; Female bottle dendrobium candidum protocorm described in this step is meant: with the dendrobium candidum protocorm that obtains behind the dendrobium candidum protocorm successive transfer culture, successive transfer culture is meant the dendrobium candidum protocorm is transferred to continuous culture in another blake bottle from a blake bottle;
C) magnetic treatment will be by step B) the culture medium inoculated blake bottle that obtains places the magnetic field magnetic treatment, and the magnetic treatment time is 60min, and magnetic induction intensity is 12mT (milli tesla), obtains the culture medium inoculated blake bottle of magnetic treatment;
D) enrichment culture; Will be by step C) the culture medium inoculated blake bottle of the magnetic treatment that obtains places culturing room's enrichment culture; Culturing room's temperature is 24 ℃ of daytimes, and at 20 ℃ of nights, illuminance is 1200LUX; Light application time is 12h/ days, obtains weight propagation multiple and be 3.3 times dendrobium candidum protocorm.
Embodiment 3:
A) preparing culture medium; Preparing culture medium in blake bottle; Medium is: 1/2 MS+ NAA (naa) 0.2mg/L+ 6-BA (6-benzyladenine) 0.8mg/L+KT (kinetin) 1mg/L+ sucrose 40g/L+ agar 7.5g/L+ powder activated carbon 1.5g/L, and the pH value is 5.7, with this medium high-temperature sterilization; The concrete grammar of high-temperature sterilization is: medium is placed on is warming up to 120 ℃ (pressure is 0.1MPa) in the high-pressure sterilizing pot; And under this temperature and pressure, keep 20min, to be cooled to room temperature, obtain filling the blake bottle of medium;
B) inoculation is inoculated in female bottle dendrobium candidum protocorm 0.5g by steps A) culture vessel that fills medium that obtains is in the blake bottle, obtains the culture medium inoculated blake bottle, the female bottle dendrobium candidum protocorm described in this step is with the description to embodiment 1;
C) magnetic treatment will be by step B) the culture medium inoculated blake bottle that obtains places the magnetic field magnetic treatment, and the magnetic treatment time is 300min, and magnetic induction intensity is 6mT (milli tesla), obtains the culture medium inoculated blake bottle of magnetic treatment;
D) enrichment culture; Will be by step C) the culture medium inoculated blake bottle of the magnetic treatment that obtains places culturing room's enrichment culture; Culturing room's temperature is 24 ℃ of daytimes, and at 21 ℃ of nights, illuminance is 1300LUX; Light application time is 12h/ days, obtains weight propagation multiple and be 5.8 times dendrobium candidum protocorm.
Embodiment 4:
A) preparing culture medium; Preparing culture medium in blake bottle, medium is: 1/2 MS+ NAA (naa) 0.3mg/L+ 6-BA (6-benzyladenine) 1mg/L+KT (kinetin) 1mg/L+ sucrose, 40 g/L+ agar 8.5g/L+ powder activated carbon 3g/L, the pH value is 5.7; With this medium high-temperature sterilization; The concrete grammar of high-temperature sterilization is: medium is placed on is warming up to 120 ℃ in the high-pressure sterilizing pot, pressure is 0.07MPa, and under this temperature and pressure, keeps 22min; To be cooled to room temperature, obtain filling the blake bottle of medium;
B) inoculation is inoculated in female bottle dendrobium candidum protocorm 0.5g by steps A) culture vessel that fills medium that obtains is in the blake bottle, obtains the culture medium inoculated blake bottle, the female bottle dendrobium candidum protocorm described in this step is with the description to embodiment 1;
C) magnetic treatment will be by step B) the culture medium inoculated blake bottle that obtains places the magnetic field magnetic treatment, and the magnetic treatment time is 300min, and magnetic induction intensity is 17mT (milli tesla), obtains the culture medium inoculated blake bottle of magnetic treatment;
D) enrichment culture; Will be by step C) the culture medium inoculated blake bottle of the magnetic treatment that obtains places culturing room's enrichment culture; Culturing room's temperature is 24 ℃ of daytimes, and at 21 ℃ of nights, illuminance is 1000LUX; Light application time is 12h/ days, obtains weight propagation multiple and be 4.9 times dendrobium candidum protocorm.
Embodiment 5:
A) preparing culture medium; Preparing culture medium in blake bottle, medium is: 1/2 MS+ NAA (naa) 0.3mg/L+ 6-BA (6-benzyladenine) 1mg/L+KT (kinetin) 1mg/L+ sucrose 30g/L+ agar 8.5g/L+ powder activated carbon 1.8g/L, the pH value is 5.7; With this medium high-temperature sterilization; The concrete grammar of high-temperature sterilization is: medium is placed on is warming up to 121 ℃ in the high-pressure sterilizing pot, pressure is 0.11MPa, and under this temperature and pressure, keeps 20min; To be cooled to room temperature, obtain filling the blake bottle of medium;
B) inoculation is inoculated in female bottle dendrobium candidum protocorm 0.5g by steps A) culture vessel that fills medium that obtains is in the blake bottle, obtains the culture medium inoculated blake bottle, the female bottle dendrobium candidum protocorm described in this step is with the description to embodiment 1;
C) magnetic treatment will be by step B) the culture medium inoculated blake bottle that obtains places the magnetic field magnetic treatment, and the magnetic treatment time is 300min, and magnetic induction intensity is 25mT (milli tesla), obtains the culture medium inoculated blake bottle of magnetic treatment;
D) enrichment culture; Will be by step C) the culture medium inoculated blake bottle of the magnetic treatment that obtains places culturing room's enrichment culture; Culturing room's temperature is 25 ℃ of daytimes, and at 22 ℃ of nights, illuminance is 1500LUX; Light application time is 12h/ days, obtains weight propagation multiple and be 6.0 times dendrobium candidum protocorm.
Embodiment 6:
A) preparing culture medium; Preparing culture medium in blake bottle, medium is: 1/2 MS+ NAA (naa) 0.2mg/L+ 6-BA (6-benzyladenine) 1mg/L+KT (kinetin) 0.8mg/L+ sucrose, 35 g/L+ agar 8g/L+ powder activated carbon 2.5g/L, the pH value is 5.7; With this medium high-temperature sterilization; The concrete grammar of high-temperature sterilization is: medium is placed on is warming up to 120 ℃ in the high-pressure sterilizing pot, pressure is 0.1MPa, and under this temperature and pressure, keeps 20min; To be cooled to room temperature, obtain filling the blake bottle of medium;
B) inoculation is inoculated in female bottle dendrobium candidum protocorm 0.5g by steps A) culture vessel that fills medium that obtains is in the blake bottle, obtains the culture medium inoculated blake bottle, the female bottle dendrobium candidum protocorm described in this step is with the description to embodiment 1;
C) magnetic treatment will be by step B) the culture medium inoculated blake bottle that obtains places the magnetic field magnetic treatment, and the magnetic treatment time is 600min, and magnetic induction intensity is 25mT (milli tesla), obtains the culture medium inoculated blake bottle of magnetic treatment;
D) enrichment culture; Will be by step C) the culture medium inoculated blake bottle of the magnetic treatment that obtains places culturing room's enrichment culture; Culturing room's temperature is 25 ℃ of daytimes, and at 22 ℃ of nights, illuminance is 1900LUX; Light application time is 12h/ days, obtains weight propagation multiple and be 7.2 times dendrobium candidum protocorm.
Embodiment 7:
A) preparing culture medium; Preparing culture medium in blake bottle, medium is: 1/2 MS+ NAA (naa) 0.3mg/L+ 6-BA (6-benzyladenine) 0.9mg/L+KT (kinetin) 1mg/L+ sucrose, 35 g/L+ agar 8g/L+ powder activated carbon 3g/L, the pH value is 5.7; With this medium high-temperature sterilization; The concrete grammar of high-temperature sterilization is: medium is placed on is warming up to 121 ℃ in the high-pressure sterilizing pot, pressure is 0.11MPa, and under this temperature and pressure, keeps 20min; To be cooled to room temperature, obtain filling the blake bottle of medium;
B) inoculation is inoculated in female bottle dendrobium candidum protocorm 0.5g by steps A) culture vessel that fills medium that obtains is in the blake bottle, obtains the culture medium inoculated blake bottle, the female bottle dendrobium candidum protocorm described in this step is with the description to embodiment 1;
C) magnetic treatment will be by step B) the culture medium inoculated blake bottle that obtains places the magnetic field magnetic treatment, and the magnetic treatment time is 900min, and magnetic induction intensity is 12mT (milli tesla), obtains the culture medium inoculated blake bottle of magnetic treatment;
D) enrichment culture; Will be by step C) the culture medium inoculated blake bottle of the magnetic treatment that obtains places culturing room's enrichment culture; Culturing room's temperature is 24 ℃ of daytimes, and at 20 ℃ of nights, illuminance is 2000LUX; Light application time is 12h/ days, obtains weight propagation multiple and be 7.5 times dendrobium candidum protocorm.
Embodiment 8:
A) preparing culture medium; Preparing culture medium in blake bottle, medium is: 1/2 MS+ NAA (naa) 0.3mg/L+ 6-BA (6-benzyladenine) 1mg/L+KT (kinetin) 1mg/L+ sucrose, 30 g/L+ agar 7.5g/L+ powder activated carbon 2g/L, the pH value is 5.7; With this medium high-temperature sterilization; The concrete grammar of high-temperature sterilization is: medium is placed on is warming up to 121 ℃ in the high-pressure sterilizing pot, pressure is 0.11MPa, and under this temperature and pressure, keeps 20min; To be cooled to room temperature, obtain filling the blake bottle of medium;
B) inoculation is inoculated in female bottle dendrobium candidum protocorm 0.5g by steps A) culture vessel that fills medium that obtains is in the blake bottle, obtains the culture medium inoculated blake bottle, the female bottle dendrobium candidum protocorm described in this step is with the description to embodiment 1;
C) magnetic treatment will be by step B) the culture medium inoculated blake bottle that obtains places the magnetic field magnetic treatment, and the magnetic treatment time is 900min, and magnetic induction intensity is 17mT (milli tesla), obtains the culture medium inoculated blake bottle of magnetic treatment;
D) enrichment culture; Will be by step C) the culture medium inoculated blake bottle of the magnetic treatment that obtains places culturing room's enrichment culture; Culturing room's temperature is 25 ℃ of daytimes, and at 21 ℃ of nights, illuminance is 1800LUX; Light application time is 12h/ days, obtains weight propagation multiple and be 6.1 times dendrobium candidum protocorm.
Embodiment 9:
A) preparing culture medium; Preparing culture medium in blake bottle, medium is: 1/2 MS+ NAA (naa) 0.3mg/L+ 6-BA (6-benzyladenine) 0.9mg/L+KT (kinetin) 1mg/L+ sucrose, 35 g/L+ agar 8g/L+ powder activated carbon 2g/L, the pH value is 5.7; With this medium high-temperature sterilization; The concrete grammar of high-temperature sterilization is: medium is placed on is warming up to 120 ℃ in the high-pressure sterilizing pot, pressure is 0.1MPa, and under this temperature and pressure, keeps 20min; To be cooled to room temperature, obtain filling the blake bottle of medium;
B) inoculation is inoculated in female bottle dendrobium candidum protocorm 0.5g by steps A) culture vessel that fills medium that obtains is in the blake bottle, obtains the culture medium inoculated blake bottle, the female bottle dendrobium candidum protocorm described in this step is with the description to embodiment 1;
C) magnetic treatment will be by step B) the culture medium inoculated blake bottle that obtains places the magnetic field magnetic treatment, and the magnetic treatment time is 900min, and magnetic induction intensity is 25mT (milli tesla), obtains the culture medium inoculated blake bottle of magnetic treatment;
D) enrichment culture; Will be by step C) the culture medium inoculated blake bottle of the magnetic treatment that obtains places culturing room's enrichment culture; Culturing room's temperature is 25 ℃ of daytimes, and at 22 ℃ of nights, illuminance is 2000LUX; Light application time is 12h/ days, obtains weight propagation multiple and be 9.1 times dendrobium candidum protocorm.
Embodiment 10:
A) preparing culture medium; Preparing culture medium in blake bottle; Medium is: 1/2 MS+ NAA (naa) 0.2mg/L+ 6-BA (6-benzyladenine) 1mg/L+KT (kinetin) 0.8mg/L+ sucrose, 35 g/L+ agar 8g/L+ powder activated carbon 2g/L, and the pH value is 5.7, with this medium high-temperature sterilization; The concrete grammar of high-temperature sterilization is: medium is placed on is warming up to 121 ℃ (pressure is 0.11MPa) in the high-pressure sterilizing pot; And under this temperature and pressure, keep 20min, to be cooled to room temperature, obtain filling the blake bottle of medium;
B) inoculation is inoculated in female bottle dendrobium candidum protocorm 0.5g by steps A) culture vessel that fills medium that obtains is in the blake bottle, obtains the culture medium inoculated blake bottle, the female bottle dendrobium candidum protocorm described in this step is with the description to embodiment 1;
C) magnetic treatment will be by step B) the culture medium inoculated blake bottle that obtains places the magnetic field magnetic treatment, and the magnetic treatment time is 600min, and magnetic induction intensity is 25mT (milli tesla), obtains the culture medium inoculated blake bottle of magnetic treatment;
D) enrichment culture; Will be by step C) the culture medium inoculated blake bottle of the magnetic treatment that obtains places culturing room's enrichment culture; Culturing room's temperature is 24 ℃ of daytimes, and at 21 ℃ of nights, illuminance is 1700LUX; Light application time is 12h/ days, obtains weight propagation multiple and be 7.2 times dendrobium candidum protocorm.
Embodiment 11:
A) preparing culture medium, preparing culture medium in blake bottle, medium is: 1/2 MS+ NAA (naa) 0.2mg/L+ 6-BA (6-benzyladenine) 1mg/L+KT (kinetin) 0.8mg/L+ tri-iron tetroxide (Fe
3
O
4
) 2g/L+sucrose 25g/L+agar 8g/L; The pH value is 5.7, and with this medium high-temperature sterilization, the concrete grammar of high-temperature sterilization is: medium is placed on is warming up to 120 ℃ in the high-pressure sterilizing pot; Pressure is 0.1MPa; And under this temperature and pressure, keep 20min, to be cooled to room temperature, obtain filling the blake bottle that medium contains tri-iron tetroxide;
B) inoculation; Female bottle dendrobium candidum protocorm 0.5g is inoculated in by steps A) obtain to fill the culture vessel that medium contains tri-iron tetroxide be in the blake bottle; Obtain the culture medium inoculated blake bottle; Mother described in this a step bottle dendrobium candidum protocorm is meant: get the capsule that dendrobium candidum does not have cracking, the use mass percent concentration is 75% ethanol wipe surfaces earlier, and putting into mass percent concentration again is the molten (HgCl of mercury chloride of 0.1%-0.2%
2
) soak 7-12min in the liquid; (aqua sterilisa is meant distilled water or ordinary tap water be placed in the high-pressure sterilizing pot temperature is risen to 115-125 ℃ to use aqua sterilisa then; Pressure is 0.07-0.135MPa), and be cooled to room temperature after keeping 20-25min) clean 3 times, 4 times or 5 times, cut capsule open; Get its embryo and embryo is seeded on the MS medium of improvement the protocorm that cultivation grew in 1-2 month;
C) magnetic treatment will be by step B) the culture medium inoculated blake bottle that obtains places the magnetic field magnetic treatment, and the magnetic treatment time is 60min, and magnetic induction intensity is 12 mT (milli teslas), obtains the culture medium inoculated blake bottle of magnetic treatment;
D) enrichment culture; Will be by step C) the culture medium inoculated blake bottle of the magnetic treatment that obtains places culturing room's enrichment culture; Culturing room's temperature is 24 ℃ of daytimes, and at 20 ℃ of nights, illuminance is 1000LUX; Light application time is 12h/ days, obtains weight propagation multiple and be 4.9 times dendrobium candidum protocorm.
Embodiment 12:
A) preparing culture medium, preparing culture medium in blake bottle, medium is: 1/2 MS+ NAA (naa) 0.2mg/L+ 6-BA (6-benzyladenine) 0.9mg/L+KT (kinetin) 1mg/L+ tri-iron tetroxide (Fe
3
O
4
) 2g/L+sucrose 30g/L+agar 8g/L; The pH value is 5.7; With this medium high-temperature sterilization, the concrete grammar of high-temperature sterilization is: medium is placed on is warming up to 120 ℃ (pressure is 0.1MPa) in the high-pressure sterilizing pot, and under this temperature and pressure, keep 20min; To be cooled to room temperature, obtain filling the blake bottle that medium contains tri-iron tetroxide;
B) inoculation; Female bottle dendrobium candidum protocorm 0.5g is inoculated in by steps A) obtain to fill the culture vessel that medium contains tri-iron tetroxide be in the blake bottle; Obtain the culture medium inoculated blake bottle, the female bottle dendrobium candidum protocorm described in this step is with the description to embodiment 11;
C) magnetic treatment will be by step B) the culture medium inoculated blake bottle that obtains places the magnetic field magnetic treatment, and the magnetic treatment time is 300min, and magnetic induction intensity is 6mT (milli tesla), obtains the culture medium inoculated blake bottle of magnetic treatment;
D) enrichment culture; Will be by step C) the culture medium inoculated blake bottle of the magnetic treatment that obtains places culturing room's enrichment culture; Culturing room's temperature is 25 ℃ of daytimes, and at 22 ℃ of nights, illuminance is 2000LUX; Light application time is 12h/ days, obtains weight propagation multiple and be 7.3 times dendrobium candidum protocorm.
Embodiment 13:
A) preparing culture medium, preparing culture medium in blake bottle, medium is: 1/2 MS+ NAA (naa) 0.2mg/L+ 6-BA (6-benzyladenine) 1mg/L+KT (kinetin) 1mg/L+ tri-iron tetroxide (Fe
3
O
4
) 2g/L+sucrose 30g/L+agar 8.5g/L; The pH value is 5.8, and with this medium high-temperature sterilization, the concrete grammar of high-temperature sterilization is: medium is placed on is warming up to 120 ℃ in the high-pressure sterilizing pot; Pressure is 0.1MP; And under this temperature and pressure, keep 20min, to be cooled to room temperature, obtain filling the blake bottle that medium contains tri-iron tetroxide;
B) inoculation; Female bottle dendrobium candidum protocorm 0.5g is inoculated in by steps A) obtain to fill the culture vessel that medium contains tri-iron tetroxide be in the blake bottle; Obtain the culture medium inoculated blake bottle, the female bottle dendrobium candidum protocorm described in this step is with the description to embodiment 11;
C) magnetic treatment will be by step B) the culture medium inoculated blake bottle that obtains places the magnetic field magnetic treatment, and the magnetic treatment time is 300min, and magnetic induction intensity is 12mT (milli tesla), obtains the culture medium inoculated blake bottle of magnetic treatment;
D) enrichment culture; Will be by step C) the culture medium inoculated blake bottle of the magnetic treatment that obtains places culturing room's enrichment culture; Culturing room's temperature is 24 ℃ of daytimes, and at 20 ℃ of nights, illuminance is 1300LUX; Light application time is 12h/ days, obtains weight propagation multiple and be 5.6 times dendrobium candidum protocorm.
Embodiment 14:
A) preparing culture medium, preparing culture medium in blake bottle, medium is: 1/2 MS+ NAA (naa) 0.3mg/L+ 6-BA (6-benzyladenine) 1mg/L+KT (kinetin) 1mg/L+ tri-iron tetroxide (Fe
3
O
4
) 2g/L+sucrose 40g/L+agar 8.5g/L; The pH value is 5.8, and with this medium high-temperature sterilization, the concrete grammar of high-temperature sterilization is: medium is placed on is warming up to 121 ℃ in the high-pressure sterilizing pot; Pressure is 0.11MPa; And under this temperature and pressure, keep 20min, to be cooled to room temperature, obtain filling the blake bottle that medium contains tri-iron tetroxide;
B) inoculation; Female bottle dendrobium candidum protocorm 0.5g is inoculated in by steps A) obtain to fill the culture vessel that medium contains tri-iron tetroxide be in the blake bottle; Obtain the culture medium inoculated blake bottle, the female bottle dendrobium candidum protocorm described in this step is with the description to embodiment 11;
C) magnetic treatment will be by step B) the culture medium inoculated blake bottle that obtains places the magnetic field magnetic treatment, and the magnetic treatment time is 300min, and magnetic induction intensity is 17mT (milli tesla), obtains the culture medium inoculated blake bottle of magnetic treatment;
D) enrichment culture; Will be by step C) the culture medium inoculated blake bottle of the magnetic treatment that obtains places culturing room's enrichment culture; Culturing room's temperature is 24 ℃ of daytimes, and at 21 ℃ of nights, illuminance is 1600LUX; Light application time is 12h/ days, obtains weight propagation multiple and be 6.1 times dendrobium candidum protocorm.
Embodiment 14:
A) preparing culture medium, preparing culture medium in blake bottle, medium is: 1/2 MS+ NAA (naa) 0.3mg/L+ 6-BA (6-benzyladenine) 1mg/L+KT (kinetin) 1mg/L+ tri-iron tetroxide (Fe
3
O
4
) 2g/L+sucrose 40g/L+agar 8.5g/L; The pH value is 5.8, and with this medium high-temperature sterilization, the concrete grammar of high-temperature sterilization is: medium is placed on is warming up to 120 ℃ in the high-pressure sterilizing pot; Pressure is 0.1MPa; And under this temperature and pressure, keep 20min, to be cooled to room temperature, obtain filling the blake bottle that medium contains tri-iron tetroxide;
B) inoculation; Female bottle dendrobium candidum protocorm 0.5g is inoculated in by steps A) obtain to fill the culture vessel that medium contains tri-iron tetroxide be in the blake bottle; Obtain the culture medium inoculated blake bottle, the female bottle dendrobium candidum protocorm described in this step is with the description to embodiment 11;
C) magnetic treatment will be by step B) the culture medium inoculated blake bottle that obtains places the magnetic field magnetic treatment, and the magnetic treatment time is 600min, and magnetic induction intensity is 12mT (milli tesla), obtains the culture medium inoculated blake bottle of magnetic treatment;
D) enrichment culture; Will be by step C) the culture medium inoculated blake bottle of the magnetic treatment that obtains places culturing room's enrichment culture; Culturing room's temperature is 24 ℃ of daytimes, and at 22 ℃ of nights, illuminance is 1700LUX; Light application time is 12h/ days, obtains weight propagation multiple and be 5.9 times dendrobium candidum protocorm.
Embodiment 15:
A) preparing culture medium, preparing culture medium in blake bottle, medium is: 1/2 MS+ NAA (naa) 0.2mg/L+ 6-BA (6-benzyladenine) 1mg/L+KT (kinetin) 1mg/L+ tri-iron tetroxide (Fe
3
O
4
) 4g/L+sucrose 20g/L+agar 8.5g/L; The pH value is 5.8, and with this medium high-temperature sterilization, the concrete grammar of high-temperature sterilization is: medium is placed on is warming up to 120 ℃ in the high-pressure sterilizing pot; Pressure is 0.1MPa; And under this temperature and pressure, keep 20min, to be cooled to room temperature, obtain filling the blake bottle that medium contains tri-iron tetroxide;
B) inoculation; Female bottle dendrobium candidum protocorm 0.5g is inoculated in by steps A) obtain to fill the culture vessel that medium contains tri-iron tetroxide be in the blake bottle; Obtain the culture medium inoculated blake bottle, the female bottle dendrobium candidum protocorm described in this step is with the description to embodiment 11;
C) magnetic treatment will be by step B) the culture medium inoculated blake bottle that obtains places the magnetic field magnetic treatment, and the magnetic treatment time is 60min, and magnetic induction intensity is 25mT (milli tesla), obtains the culture medium inoculated blake bottle of magnetic treatment;
D) enrichment culture; Will be by step C) the culture medium inoculated blake bottle of the magnetic treatment that obtains places culturing room's enrichment culture; Culturing room's temperature is 25 ℃ of daytimes, and at 22 ℃ of nights, illuminance is 2000LUX; Light application time is 12h/ days, obtains weight propagation multiple and be 3.5 times dendrobium candidum protocorm.
Embodiment 16:
A) preparing culture medium, preparing culture medium in blake bottle, medium is: 1/2 MS+ NAA (naa) 0.2mg/L+ 6-BA (6-benzyladenine) 1mg/L+KT (kinetin) 1mg/L+ tri-iron tetroxide (Fe
3
O
4
) 4g/L+sucrose 20g/L+agar 8.5g/L; The pH value is 5.8, and with this medium high-temperature sterilization, the concrete grammar of high-temperature sterilization is: medium is placed on is warming up to 121 ℃ in the high-pressure sterilizing pot; Pressure is 0.11MPa; And under this temperature and pressure, keep 20min, to be cooled to room temperature, obtain filling the blake bottle that medium contains tri-iron tetroxide;
B) inoculation; Female bottle dendrobium candidum protocorm 0.5g is inoculated in by steps A) obtain to fill the culture vessel that medium contains tri-iron tetroxide be in the blake bottle; Obtain the culture medium inoculated blake bottle, the female bottle dendrobium candidum protocorm described in this step is with the description to embodiment 11;
C) magnetic treatment will be by step B) the culture medium inoculated blake bottle that obtains places the magnetic field magnetic treatment, and the magnetic treatment time is 300min, and magnetic induction intensity is 25mT (milli tesla), obtains the culture medium inoculated blake bottle of magnetic treatment;
D) enrichment culture; Will be by step C) the culture medium inoculated blake bottle of the magnetic treatment that obtains places culturing room's enrichment culture; Culturing room's temperature is 24 ℃ of daytimes, and at 22 ℃ of nights, illuminance is 1750LUX; Light application time is 12h/ days, obtains weight propagation multiple and be 4.0 times dendrobium candidum protocorm.
Embodiment 17:
A) preparing culture medium, preparing culture medium in blake bottle, medium is: 1/2 MS+ NAA (naa) 0.2mg/L+ 6-BA (6-benzyladenine) 1mg/L+KT (kinetin) 1mg/L+ tri-iron tetroxide (Fe
3
O
4
) 4g/L+sucrose 20g/L+agar 8.5g/L; The pH value is 5.8, and with this medium high-temperature sterilization, the concrete grammar of high-temperature sterilization is: medium is placed on is warming up to 120 ℃ in the high-pressure sterilizing pot; Pressure is 0.1MPa; And under this temperature and pressure, keep 20min, to be cooled to room temperature, obtain filling the blake bottle that medium contains tri-iron tetroxide;
B) inoculation; Female bottle dendrobium candidum protocorm 0.5g is inoculated in by steps A) obtain to fill the culture vessel that medium contains tri-iron tetroxide be in the blake bottle; Obtain the culture medium inoculated blake bottle, the female bottle dendrobium candidum protocorm described in this step is with the description to embodiment 11;
C) magnetic treatment will be by step B) the culture medium inoculated blake bottle that obtains places the magnetic field magnetic treatment, and the magnetic treatment time is 600min, and magnetic induction intensity is 17mT (milli tesla), obtains the culture medium inoculated blake bottle of magnetic treatment;
D) enrichment culture; Will be by step C) the culture medium inoculated blake bottle of the magnetic treatment that obtains places culturing room's enrichment culture; Culturing room's temperature is 24 ℃ of daytimes, and at 20 ℃ of nights, illuminance is 1250LUX; Light application time is 12h/ days, obtains weight propagation multiple and be 4.5 times dendrobium candidum protocorm.
Embodiment 18:
A) preparing culture medium, preparing culture medium in blake bottle, medium is: 1/2 MS+ NAA (naa) 0.2mg/L+ 6-BA (6-benzyladenine) 1mg/L+KT (kinetin) 0.9mg/L+ tri-iron tetroxide (Fe
3
O
4
) 6g/L+sucrose 40g/L+agar 8.5g/L; The pH value is 5.8, and with this medium high-temperature sterilization, the concrete grammar of high-temperature sterilization is: medium is placed on is warming up to 120 ℃ in the high-pressure sterilizing pot; Pressure is 0.1MPa; And under this temperature and pressure, keep 20min, to be cooled to room temperature, obtain filling the blake bottle that medium contains tri-iron tetroxide;
B) inoculation; Female bottle dendrobium candidum protocorm 0.5g is inoculated in by steps A) obtain to fill the culture vessel that medium contains tri-iron tetroxide be in the blake bottle; Obtain the culture medium inoculated blake bottle, the female bottle dendrobium candidum protocorm described in this step is with the description to embodiment 11;
C) magnetic treatment will be by step B) the culture medium inoculated blake bottle that obtains places the magnetic field magnetic treatment, and the magnetic treatment time is 60min, and magnetic induction intensity is 6mT (milli tesla), obtains the culture medium inoculated blake bottle of magnetic treatment;
D) enrichment culture; Will be by step C) the culture medium inoculated blake bottle of the magnetic treatment that obtains places culturing room's enrichment culture; Culturing room's temperature is 24 ℃ of daytimes, and at 21 ℃ of nights, illuminance is 1850LUX; Light application time is 12h/ days, obtains weight propagation multiple and be 4.2 times dendrobium candidum protocorm.
Embodiment 19:
A) preparing culture medium, preparing culture medium in blake bottle, medium is: 1/2 MS+ NAA (naa) 0.3mg/L+ 6-BA (6-benzyladenine) 1mg/L+KT (kinetin) 1mg/L+ tri-iron tetroxide (Fe
3
O
4
) 6g/L+sucrose 30g/L+agar 9g/L; The pH value is 5.8, and with this medium high-temperature sterilization, the concrete grammar of high-temperature sterilization is: medium is placed on is warming up to 125 ℃ in the high-pressure sterilizing pot; Pressure is 0.135MPa; And under this temperature and pressure, keep 20min, to be cooled to room temperature, obtain filling the blake bottle that medium contains tri-iron tetroxide;
B) inoculation; Female bottle dendrobium candidum protocorm 0.5g is inoculated in by steps A) obtain to fill the culture vessel that medium contains tri-iron tetroxide be in the blake bottle; Obtain the culture medium inoculated blake bottle, the female bottle dendrobium candidum protocorm described in this step is with the description to embodiment 11;
C) magnetic treatment will be by step B) the culture medium inoculated blake bottle that obtains places the magnetic field magnetic treatment, and the magnetic treatment time is 60min, and magnetic induction intensity is 12mT (milli tesla), obtains the culture medium inoculated blake bottle of magnetic treatment;
D) enrichment culture; Will be by step C) the culture medium inoculated blake bottle of the magnetic treatment that obtains places culturing room's enrichment culture; Culturing room's temperature is 25 ℃ of daytimes, and at 22 ℃ of nights, illuminance is 2000LUX; Light application time is 12h/ days, obtains weight propagation multiple and be 8.5 times dendrobium candidum protocorm.
Embodiment 20:
A) preparing culture medium, preparing culture medium in blake bottle, medium is: 1/2 MS+ NAA (naa) 0.3mg/L+ 6-BA (6-benzyladenine) 1mg/L+KT (kinetin) 1mg/L+ tri-iron tetroxide (Fe
3
O
4
) 6g/L+sucrose 30g/L+agar 8.5g/L; The pH value is 5.8, and with this medium high-temperature sterilization, the concrete grammar of high-temperature sterilization is: medium is placed on is warming up to 121 ℃ in the high-pressure sterilizing pot; Pressure is 0.11MPa; And under this temperature and pressure, keep 20min, to be cooled to room temperature, obtain filling the blake bottle that medium contains tri-iron tetroxide;
B) inoculation; Female bottle dendrobium candidum protocorm 0.5g is inoculated in by steps A) obtain to fill the culture vessel that medium contains tri-iron tetroxide be in the blake bottle; Obtain the culture medium inoculated blake bottle, the female bottle dendrobium candidum protocorm described in this step is with the description to embodiment 11;
C) magnetic treatment will be by step B) the culture medium inoculated blake bottle that obtains places the magnetic field magnetic treatment, and the magnetic treatment time is 60min, and magnetic induction intensity is 17mT (milli tesla), obtains the culture medium inoculated blake bottle of magnetic treatment;
D) enrichment culture; Will be by step C) the culture medium inoculated blake bottle of the magnetic treatment that obtains places culturing room's enrichment culture; Culturing room's temperature is 25 ℃ of daytimes, and at 23 ℃ of nights, illuminance is 2000LUX; Light application time is 12h/ days, obtains weight propagation multiple and be 5.6 times dendrobium candidum protocorm.
Embodiment 21:
A) preparing culture medium, preparing culture medium in blake bottle, medium is: 1/2 MS+ NAA (naa) 0.3mg/L+ 6-BA (6-benzyladenine) 1mg/L+KT (kinetin) 1mg/L+ tri-iron tetroxide (Fe
3
O
4
) 6g/L+sucrose 30g/L+agar 8.5g/L; The pH value is 5.8, and with this medium high-temperature sterilization, the concrete grammar of high-temperature sterilization is: medium is placed on is warming up to 120 ℃ in the high-pressure sterilizing pot; Pressure is 0.1MPa; And under this temperature and pressure, keep 20min, to be cooled to room temperature, obtain filling the blake bottle that medium contains tri-iron tetroxide;
B) inoculation; Female bottle dendrobium candidum protocorm 0.5g is inoculated in by steps A) obtain to fill the culture vessel that medium contains tri-iron tetroxide be in the blake bottle; Obtain the culture medium inoculated blake bottle, the female bottle dendrobium candidum protocorm described in this step is with the description to embodiment 11;
C) magnetic treatment will be by step B) the culture medium inoculated blake bottle that obtains places the magnetic field magnetic treatment, and the magnetic treatment time is 300min, and magnetic induction intensity is 6mT (milli tesla), obtains the culture medium inoculated blake bottle of magnetic treatment;
D) enrichment culture; Will be by step C) the culture medium inoculated blake bottle of the magnetic treatment that obtains places culturing room's enrichment culture; Culturing room's temperature is 24 ℃ of daytimes, and at 21 ℃ of nights, illuminance is 2000LUX; Light application time is 12h/ days, obtains weight propagation multiple and be 6.4 times dendrobium candidum protocorm.
Embodiment 22:
A) preparing culture medium, preparing culture medium in blake bottle, medium is: 1/2 MS+ NAA (naa) 0.3mg/L+ 6-BA (6-benzyladenine) 1mg/L+KT (kinetin) 1mg/L+ tri-iron tetroxide (Fe
3
O
4
) 6g/L+sucrose 30g/L+agar 8.5g/L; The pH value is 5.8, and with this medium high-temperature sterilization, the concrete grammar of high-temperature sterilization is: medium is placed on is warming up to 120 ℃ in the high-pressure sterilizing pot; Pressure is 0.1MPa; And under this temperature and pressure, keep 20min, to be cooled to room temperature, obtain filling the blake bottle that medium contains tri-iron tetroxide;
B) inoculation; Female bottle dendrobium candidum protocorm 0.5g is inoculated in by steps A) obtain to fill the culture vessel that medium contains tri-iron tetroxide be in the blake bottle; Obtain the culture medium inoculated blake bottle, the female bottle dendrobium candidum protocorm described in this step is with the description to embodiment 11;
C) magnetic treatment will be by step B) the culture medium inoculated blake bottle that obtains places the magnetic field magnetic treatment, and the magnetic treatment time is 600min, and magnetic induction intensity is 17mT (milli tesla), obtains the culture medium inoculated blake bottle of magnetic treatment;
D) enrichment culture; Will be by step C) the culture medium inoculated blake bottle of the magnetic treatment that obtains places culturing room's enrichment culture; Culturing room's temperature is 24 ℃ of daytimes, and at 20 ℃ of nights, illuminance is 1950LUX; Light application time is 12h/ days, obtains weight propagation multiple and be 4.3 times dendrobium candidum protocorm.
The computing formula of the described weight propagation of
the foregoing description 1-22 multiple is: weight propagation multiple=existing weight (g) the former implantation weight of ÷ (g), and existing weight is to adopt the resulting weight of the inventive method, former implantation weight is 0.5g.
The foregoing description 11-22 also can use the protocorm that obtains by behind the dendrobium candidum protocorm successive transfer culture, and the propagation multiple of acquisition is identical with the propagation multiple of female bottle dendrobium candidum protocorm
Claims (2)
1. one kind is improved the method that the dendrobium candidum protocorm is bred multiple, it is characterized in that may further comprise the steps:
A) preparing culture medium, preparing culture medium in culture vessel, and, obtain filling the culture vessel of medium with this medium high-temperature sterilization;
B) inoculation perhaps will be inoculated in female bottle dendrobium candidum protocorm 0.5g in the culture vessel that fills medium by the protocorm 0.5g that obtains behind the dendrobium candidum protocorm successive transfer culture, obtain the culture medium inoculated culture vessel;
C) magnetic treatment places the magnetic field magnetic treatment with the culture medium inoculated culture vessel, controls magnetic treatment time and magnetic induction intensity, obtains the culture medium inoculated culture vessel of magnetic treatment;
D) enrichment culture; The culture medium inoculated culture vessel of magnetic treatment is placed culturing room's enrichment culture; And cultivation temperature, illuminance and the light application time of control culturing room; Obtaining weight propagation multiple is 3-9 dendrobium candidum protocorm doubly, and described medium is: 1/2 MS+NAA0.2-0.4mg/L+6-BA 0.8-1 mg/L+KT 0.8-1 mg/L+ sucrose 20-40g/L+ agar 7-9g/L+ powder activated carbon 1-3g/L, pH5.6-5.8; Described high-temperature sterilization is said medium to be placed in the high-pressure sterilizing pot temperature is risen to 115-125 ℃; Pressure is 0.07-0.135MPa; And be cooled to room temperature after keeping 20-25min, described female bottle dendrobium candidum protocorm is to get the capsule that dendrobium candidum does not have cracking, and using mass percent concentration earlier is 75% ethanol wipe surfaces; The mercuric chloride solution of putting into mass percent concentration again and be 0.1%-0.2% soaks 7-12min; Clean with aqua sterilisa then, then cut capsule open, get its embryo and embryo is seeded on the MS medium of improvement and cultivate until the protocorm that grows; The MS medium of improvement is meant in the MS medium NH
4NO
3Use reduces by half; Described aqua sterilisa is meant distilled water or ordinary tap water placed and is warming up to 115-125 ℃ in the high-pressure sterilizing pot that pressure is 0.07-0.135MPa, and keeps 20-25min, again through being cooled to the resulting water of room temperature; The number of times of described cleaning is 3 times, 4 times or 5 times; The described control magnetic treatment time is that the magnetic treatment time is controlled to be 60-900min; Described control magnetic induction intensity is that magnetic induction intensity is controlled to be 6-25mT, and the time of enrichment culture is 30-60 days in the described culturing room; The described cultivation temperature of controlling culturing room is that temperature is controlled to be 20-25 ℃, and described control illuminance is that illuminance is controlled to be 1000-2000LUX, and described control light application time is that light application time is controlled to be 12h/ days.
2. one kind is improved the method that the dendrobium candidum protocorm is bred multiple, it is characterized in that may further comprise the steps:
A) preparing culture medium, preparing culture medium in culture vessel, and, obtain filling the culture vessel of the medium that contains tri-iron tetroxide with this medium high-temperature sterilization;
B) inoculation; Female bottle dendrobium candidum protocorm 0.5g perhaps will be inoculated in the culture vessel that fills the medium that contains tri-iron tetroxide by the protocorm 0.5g that obtains behind the dendrobium candidum protocorm successive transfer culture, obtain containing the culture medium inoculated culture vessel of tri-iron tetroxide;
C) magnetic treatment, the culture medium inoculated culture vessel that will contain tri-iron tetroxide places the magnetic field magnetic treatment, controls magnetic treatment time and magnetic induction intensity, obtains the culture medium inoculated culture vessel that contains tri-iron tetroxide of magnetic treatment;
D) enrichment culture; The culture medium inoculated culture vessel that contains tri-iron tetroxide of magnetic treatment is placed culturing room's enrichment culture; And cultivation temperature, illuminance and the light application time of control culturing room; Obtaining weight propagation multiple is 3-8.5 dendrobium candidum protocorm doubly, and described medium is: 1/2 MS+NAA0.2-0.4mg/L+6-BA 0.8-1 mg/L+KT 0.8-1 mg/L+ tri-iron tetroxide (Fe
3O
4) 2-6g/L+sucrose 20-40g/L+ agar 7-9g/L, pH5.6-5.8; Described high-temperature sterilization is said medium to be placed in the high-pressure sterilizing pot temperature is risen to 115-125 ℃; Pressure is 0.07-0.135MPa; And be cooled to room temperature after keeping 20-25min, described female bottle dendrobium candidum protocorm is to get the capsule that dendrobium candidum does not have cracking, and using mass percent concentration earlier is 75% ethanol wipe surfaces; The mercuric chloride solution of putting into mass percent concentration again and be 0.1%-0.2% soaks 7-12min; Clean with aqua sterilisa then, then cut capsule open, get its embryo and embryo is seeded on the MS medium of improvement and cultivate until the protocorm that grows; The MS medium of improvement is meant in the MS medium NH
4NO
3Use reduces by half; Described aqua sterilisa is meant distilled water or ordinary tap water placed and is warming up to 115-125 ℃ in the high-pressure sterilizing pot that pressure is 0.07-0.135MPa, and keeps 20-25min, again through being cooled to the resulting water of room temperature; The number of times of described cleaning is 3 times, 4 times or 5 times; The described control magnetic treatment time is that the magnetic treatment time is controlled to be 60-900min; Described control magnetic induction intensity is that magnetic induction intensity is controlled to be 6-25mT, and the time of enrichment culture is 30-50 days in the described culturing room; The described cultivation temperature of controlling culturing room is that temperature is controlled to be 20-25 ℃, and described control illuminance is that illuminance is controlled to be 1000-2000LUX, and described control light application time is that light application time is controlled to be 12h/ days.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011102004982A CN102265788B (en) | 2011-07-18 | 2011-07-18 | Method for increasing proliferation multiple of dendrobium candidum protocorms |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011102004982A CN102265788B (en) | 2011-07-18 | 2011-07-18 | Method for increasing proliferation multiple of dendrobium candidum protocorms |
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| CN101715733A (en) * | 2010-01-19 | 2010-06-02 | 烟台汇鹏生物科技有限公司 | Tissue culturing method of protocorms of dendrobium candidum |
| CN101878736A (en) * | 2010-06-18 | 2010-11-10 | 沈阳药科大学 | Method for producing dendrobium candicum |
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| CN101715733A (en) * | 2010-01-19 | 2010-06-02 | 烟台汇鹏生物科技有限公司 | Tissue culturing method of protocorms of dendrobium candidum |
| CN101878736A (en) * | 2010-06-18 | 2010-11-10 | 沈阳药科大学 | Method for producing dendrobium candicum |
Non-Patent Citations (2)
| Title |
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| 徐忠传等.磁场对乌药试管苗生长的生物学效应研究.《安徽农业科学》.2008,第36卷(第23期), * |
| 蒋彧等.磁处理对春剑‘隆昌素’根状茎分化的影响研究.《观赏园艺与西部发展——中国园艺学会观赏园艺专业委员会2010年全国学术年会》.2010, * |
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