CN105613296A - Tissue culture inoculating method for dendrobium officinale - Google Patents
Tissue culture inoculating method for dendrobium officinale Download PDFInfo
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- CN105613296A CN105613296A CN201610046856.1A CN201610046856A CN105613296A CN 105613296 A CN105613296 A CN 105613296A CN 201610046856 A CN201610046856 A CN 201610046856A CN 105613296 A CN105613296 A CN 105613296A
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- 238000000034 method Methods 0.000 title claims abstract description 31
- 241001076416 Dendrobium tosaense Species 0.000 title abstract description 12
- 210000001161 mammalian embryo Anatomy 0.000 claims abstract description 52
- 238000011081 inoculation Methods 0.000 claims abstract description 27
- 238000002360 preparation method Methods 0.000 claims abstract description 18
- 239000000654 additive Substances 0.000 claims abstract description 8
- 230000000996 additive effect Effects 0.000 claims abstract description 8
- 239000007788 liquid Substances 0.000 claims abstract description 6
- 238000009331 sowing Methods 0.000 claims abstract description 6
- 238000012258 culturing Methods 0.000 claims description 18
- 239000006185 dispersion Substances 0.000 claims description 17
- 235000013399 edible fruits Nutrition 0.000 claims description 16
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 16
- 239000001963 growth medium Substances 0.000 claims description 14
- 239000007640 basal medium Substances 0.000 claims description 12
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 10
- 230000003203 everyday effect Effects 0.000 claims description 10
- 238000005286 illumination Methods 0.000 claims description 10
- 230000001954 sterilising effect Effects 0.000 claims description 10
- PRPINYUDVPFIRX-UHFFFAOYSA-N 1-naphthaleneacetic acid Chemical compound C1=CC=C2C(CC(=O)O)=CC=CC2=C1 PRPINYUDVPFIRX-UHFFFAOYSA-N 0.000 claims description 8
- CGIDKJRJBMFXKV-UHFFFAOYSA-N 6-n'-benzylpurine-6,6-diamine Chemical compound N1=CN=C2N=CN=C2C1(N)NCC1=CC=CC=C1 CGIDKJRJBMFXKV-UHFFFAOYSA-N 0.000 claims description 8
- 229920001817 Agar Polymers 0.000 claims description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 5
- 235000007164 Oryza sativa Nutrition 0.000 claims description 5
- 229930006000 Sucrose Natural products 0.000 claims description 5
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 5
- 239000008272 agar Substances 0.000 claims description 5
- 235000020415 coconut juice Nutrition 0.000 claims description 5
- 238000001816 cooling Methods 0.000 claims description 5
- 230000000249 desinfective effect Effects 0.000 claims description 5
- 239000002054 inoculum Substances 0.000 claims description 5
- 230000035800 maturation Effects 0.000 claims description 5
- 235000009566 rice Nutrition 0.000 claims description 5
- 230000007480 spreading Effects 0.000 claims description 5
- 238000003892 spreading Methods 0.000 claims description 5
- 238000003756 stirring Methods 0.000 claims description 5
- 239000005720 sucrose Substances 0.000 claims description 5
- 238000007865 diluting Methods 0.000 claims description 3
- 230000035784 germination Effects 0.000 claims description 3
- 239000013589 supplement Substances 0.000 claims description 3
- 240000007594 Oryza sativa Species 0.000 claims 1
- 230000004083 survival effect Effects 0.000 abstract description 8
- 230000008569 process Effects 0.000 abstract description 5
- 238000004519 manufacturing process Methods 0.000 abstract description 3
- 239000000126 substance Substances 0.000 abstract description 2
- 210000002257 embryonic structure Anatomy 0.000 abstract 3
- 239000012883 rooting culture medium Substances 0.000 abstract 3
- 230000000844 anti-bacterial effect Effects 0.000 abstract 1
- 238000007796 conventional method Methods 0.000 abstract 1
- 230000006698 induction Effects 0.000 abstract 1
- 241000209094 Oryza Species 0.000 description 4
- 230000004075 alteration Effects 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 241001523681 Dendrobium Species 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- SQUHHTBVTRBESD-UHFFFAOYSA-N Hexa-Ac-myo-Inositol Natural products CC(=O)OC1C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C1OC(C)=O SQUHHTBVTRBESD-UHFFFAOYSA-N 0.000 description 1
- NIPNSKYNPDTRPC-UHFFFAOYSA-N N-[2-oxo-2-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)ethyl]-2-[[3-(trifluoromethoxy)phenyl]methylamino]pyrimidine-5-carboxamide Chemical compound O=C(CNC(=O)C=1C=NC(=NC=1)NCC1=CC(=CC=C1)OC(F)(F)F)N1CC2=C(CC1)NN=N2 NIPNSKYNPDTRPC-UHFFFAOYSA-N 0.000 description 1
- 230000003385 bacteriostatic effect Effects 0.000 description 1
- 238000010923 batch production Methods 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 208000006278 hypochromic anemia Diseases 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- CDAISMWEOUEBRE-GPIVLXJGSA-N inositol Chemical compound O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](O)[C@@H]1O CDAISMWEOUEBRE-GPIVLXJGSA-N 0.000 description 1
- 229960000367 inositol Drugs 0.000 description 1
- 150000002505 iron Chemical class 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000010899 nucleation Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- CDAISMWEOUEBRE-UHFFFAOYSA-N scyllo-inosotol Natural products OC1C(O)C(O)C(O)C(O)C1O CDAISMWEOUEBRE-UHFFFAOYSA-N 0.000 description 1
- 239000011573 trace mineral Substances 0.000 description 1
- 235000013619 trace mineral Nutrition 0.000 description 1
- 238000004383 yellowing Methods 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
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- Life Sciences & Earth Sciences (AREA)
- Developmental Biology & Embryology (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- Environmental Sciences (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention discloses a tissue culture inoculating method for dendrobium officinale. The tissue culture inoculating method for the dendrobium officinale comprises the following steps: selection of dendrobium officinale pods, sowing of seed embryos, preparation of sterile seed embryo mixed liquid, preparation of a germinating and rooting culture medium, and inoculation and culture of the seed embryos. The method disclosed by the invention is a simple and effective tissue culture inoculating method. According to the method, the germinating and rooting culture medium is inoculated to the cultured seed embryos through once induction, so that repeated culture rotation is not required; moreover, the germinating and rooting culture medium prepared in the method disclosed by the invention is mainly based on a natural antibacterial additive, and does not have harmful chemical component additive, so that the problems that the debugging time is long, the workload is long, the pollution rate is high, and the stability of produced seed seedlings is poor in conventional artificial one-by-one inoculation can be effectively solved; the pollution rate in the tissue culture process of the dendrobium officinale can be greatly reduced, and the tissue culture cost of the dendrobium officinale can be reduced; the tissue culture inoculating efficiency and the transplanting survival rate of the dendrobium officinale are greatly improved; the production efficiency is 3 to 4 times that of the conventional method; the transplanting survival rate can reach more than or equal to 92 to 93 percent.
Description
Technical field
The invention belongs to plant biotechnology field, be specifically related to a kind of candidum tissue culturing inoculation method.
Background technology
Herba Dendrobii (Dendrobiumofficinale) for the orchid family Dendrobium herbaceos perennial, dendrobium officinale is mainly distributed on the ground such as Zhejiang, Anhui, Jiangxi, Guangxi. The seed structure uniqueness of Herba Dendrobii, without endosperm, natural breed rate is extremely low, and the madness of wild resource is excavated by people in addition, and dendrobium officinale resource is endangered. Along with the development of modern biotechnology tissue culture technology, the artificial breeding technology of present seedlings of Dendrobium officinale is ripe gradually. The seedlings of Dendrobium officinale of current batch production is bred, being generally adopted Herba Dendrobii fruit pod is kind of a source, through artificial seeding's culture medium, after making embryo germination, it is about the 8-10 cultivation of individual month can obtain Herba Dendrobii finished product tissue cultured seedling through wooden dipper kind, single Seedling switching, strong sprout, four-stage of taking root. In single Seedling switching, strong sprout, take root in three phases switching process, artificial inoculation tweezers are needed frequently to be transferred to one by one in new culture medium by seedling, needing to expend substantial amounts of labour force and raw material in switching process, operation is loaded down with trivial details, inefficiency, causes that production cost remains high. Additionally in switching process, if dynamics control is improper, Seedling body physical damnification can be caused when gripping seedling, cause the situations such as Seedling bulk-growth is slow, yellowing leaf, overall chlorosis are dead, have a strong impact on the survival rate of seedling.
Summary of the invention
The technical problem to be solved is, for the deficiencies in the prior art, it is provided that a kind of simple, efficient candidum tissue culturing inoculation method.
This invention address that the technical scheme that above-mentioned technical problem adopts is: a kind of candidum tissue culturing inoculation method, comprise the following steps:
1) selection of Herba Dendrobii fruit pod: the Herba Dendrobii selecting outdoor pseudo-wild cultivating 3-6 raw is tied and grown the maturation formed 110-130 days and do not ftracture really pod;
2) embryo sowing: by Herba Dendrobii fruit pod on superclean bench after the alcohol disinfecting 12-15min that mass percent concentration is 65-75%, then the embryo in fruit pod is seeded in sterile beaker;
3) preparation of aseptic embryo mixed liquor: in step 2) sterile beaker in add sterilized water stirring, embryo is dispersed in sterilized water, dispersion is 60-100/mL, obtains aseptic embryo mixed liquor;
4) preparation of root media is sprouted: by culture medium based on 1/2MS culture medium, stereometer with this basal medium, following additive is added: naphthalene acetic acid 0.7-0.9mg/L in this basal medium, activated carbon 0.2-0.5g/L, 6-benzyl aminoadenine 0.05-0.2mg/L, coconut juice 50-80g/L, peeling Fructus Musae 30-50g/L, rice 20-30g/L, sucrose 25-35g/L and agar 5.8-6.3g/L, obtain sprouting root media, and the pH value of this sprouting root media is adjusted to 6.0 ~ 6.2, afterwards the root media of sprouting of preparation is dispensed into in the 650mL tissue culture bottle of air-vent, the liquid filling degree of depth of each tissue culture bottle is 3-4cm, then by each tissue culture bottle in high-pressure sterilizing pot in 121 DEG C of sterilizing 20-23min, after cooling standby,
5) inoculation of embryo and cultivation: being inoculated into by aseptic embryo mixed liquor sterile pipette in sprouting root media, the inoculum concentration of each tissue culture bottle is 800-1000 �� L, then be coated with aseptic spreading rod and put down, by the bottle cap screwing of tissue culture bottle, inoculation completes; Afterwards by each tissue culture bottle dark treatment 5-7 days at 22-24 DEG C, then every day intensity of illumination be 1200-1500Lux, light application time be 10-11h culture environment under cultivate 30-40 days, last every day intensity of illumination be 2500-3000Lux, light application time be 12-13h culture environment under cultivate some skies, until embryo germination obtain candidum tissue culturing seedling.
MS basal medium is currently used most common culture medium, its composition includes a great number of elements, trace element, iron salt, organic principle, inositol etc., 1/2MS basal medium and a great number of elements become the culture medium of original 1/2, it is possible to self-control or selection commercially available prod.
Preferably, in step 3), the defining method of the dispersion of embryo is: pipette the aseptic embryo mixed liquor of 1mL with liquid-transfering gun, after diluting 10 times with sterilized water, embryo is counted, it is determined that its dispersion, if dispersion is unsatisfactory for the requirement of 60-100/mL, in sterile beaker, then supplement embryo or sterilized water, until dispersion meets requirement.
Compared with prior art, it is an advantage of the current invention that: candidum tissue culturing inoculation method of the present invention, it is a kind of simple, efficient group training inoculation method, the method is inoculated in cultivation embryo through once inducing sprouting root media, it is made without switching repeatedly, and the sprouting root media of the inventive method preparation is based on natural bacteriostatic additive, add without harmful chemical component, it is thus possible to it is long by an inoculation debugging time effectively to solve Traditional Man, workload is big, pollution rate is high, the problem of the seedling poor stability produced, the inventive method can be greatly reduced the pollution rate in candidum tissue culturing process, reduce the group training cost of Herba Dendrobii, candidum tissue culturing inoculation method of the present invention makes candidum tissue culturing inoculation efficiency and transplanting survival rate be greatly improved, and production efficiency is 3-4 times of traditional method, and transplanting survival rate is up to more than 92-93%.
Detailed description of the invention
Below in conjunction with embodiment, the present invention is described in further detail.
The candidum tissue culturing inoculation method of embodiment 1, comprises the following steps:
1) selection of Herba Dendrobii fruit pod: select the Herba Dendrobii of outdoor pseudo-wild cultivating life in 3 years to be tied and grow the maturation formed 110 days not ftracture really pod;
2) embryo sowing: by Herba Dendrobii fruit pod on superclean bench after the alcohol disinfecting 12min that mass percent concentration is 65%, then the embryo in fruit pod is seeded in sterile beaker;
3) preparation of aseptic embryo mixed liquor: in step 2) sterile beaker in add sterilized water stirring, embryo is dispersed in sterilized water, dispersion is 60-70/mL, obtains aseptic embryo mixed liquor;
4) preparation of root media is sprouted: by culture medium based on 1/2MS culture medium, stereometer with this basal medium, following additive is added: naphthalene acetic acid (NAA) 0.75mg/L in this basal medium, activated carbon 0.3g/L, 6-benzyl aminoadenine (6-BA) 0.08mg/L, coconut juice 50g/L, peeling Fructus Musae 35g/L, rice 22g/L, sucrose 25g/L and agar 5.8g/L, obtain sprouting root media, and the pH value of this sprouting root media is adjusted to 6.0, afterwards the root media of sprouting of preparation is dispensed into in the 650mL tissue culture bottle of air-vent, the liquid filling degree of depth of each tissue culture bottle is 3cm, then by each tissue culture bottle in high-pressure sterilizing pot in 121 DEG C of sterilizing 20min, after cooling standby,
5) inoculation of embryo and cultivation: being inoculated into by aseptic embryo mixed liquor sterile pipette in sprouting root media, the inoculum concentration of each tissue culture bottle is 800 �� L, then be coated with aseptic spreading rod and put down, by the bottle cap screwing of tissue culture bottle, inoculation completes; Afterwards by each tissue culture bottle dark treatment 5 days at 22 DEG C, then every day intensity of illumination be 1200-1500Lux, light application time be 10-11h culture environment under cultivate 30 days, last every day intensity of illumination be 2500-3000Lux, light application time be 12-13h culture environment under cultivate 130 days after, obtain candidum tissue culturing seedling, tissue cultured seedling is evenly distributed, Seedling body healthy and strong, transplanting survival rate is 92.3%, and aberration rate is zero.
The candidum tissue culturing inoculation method of embodiment 2, comprises the following steps:
1) selection of Herba Dendrobii fruit pod: select the Herba Dendrobii of outdoor pseudo-wild cultivating life in 5 years to be tied and grow the maturation formed 120 days not ftracture really pod;
2) embryo sowing: by Herba Dendrobii fruit pod on superclean bench after the alcohol disinfecting 14min that mass percent concentration is 70%, then the embryo in fruit pod is seeded in sterile beaker;
3) preparation of aseptic embryo mixed liquor: in step 2) sterile beaker in add sterilized water stirring, embryo is dispersed in sterilized water, dispersion is 75-85/mL, obtains aseptic embryo mixed liquor;
4) preparation of root media is sprouted: by culture medium based on 1/2MS culture medium, stereometer with this basal medium, following additive is added: naphthalene acetic acid (NAA) 0.8mg/L in this basal medium, activated carbon 0.35g/L, 6-benzyl aminoadenine (6-BA) 0.075mg/L, coconut juice 65g/L, peeling Fructus Musae 40g/L, rice 25g/L, sucrose 30g/L and agar 6.0g/L, obtain sprouting root media, and the pH value of this sprouting root media is adjusted to 6.1, afterwards the root media of sprouting of preparation is dispensed into in the 650mL tissue culture bottle of air-vent, the liquid filling degree of depth of each tissue culture bottle is 3.5cm, then by each tissue culture bottle in high-pressure sterilizing pot in 121 DEG C of sterilizing 22min, after cooling standby,
5) inoculation of embryo and cultivation: being inoculated into by aseptic embryo mixed liquor sterile pipette in sprouting root media, the inoculum concentration of each tissue culture bottle is 900 �� L, then be coated with aseptic spreading rod and put down, by the bottle cap screwing of tissue culture bottle, inoculation completes; Afterwards by each tissue culture bottle dark treatment 6 days at 23 DEG C, then every day intensity of illumination be 1200-1500Lux, light application time be 10-11h culture environment under cultivate 32 days, last every day intensity of illumination be 2500-3000Lux, light application time be 12-13h culture environment under cultivate 118 days after, obtain candidum tissue culturing seedling, tissue cultured seedling is evenly distributed, Seedling body healthy and strong, transplanting survival rate is 93.2%, and aberration rate is zero.
The candidum tissue culturing inoculation method of embodiment 3, comprises the following steps:
1) selection of Herba Dendrobii fruit pod: select the Herba Dendrobii of outdoor pseudo-wild cultivating life in 6 years to be tied and grow the maturation formed 130 days not ftracture really pod;
2) embryo sowing: by Herba Dendrobii fruit pod on superclean bench after the alcohol disinfecting 14min that mass percent concentration is 75%, then the embryo in fruit pod is seeded in sterile beaker;
3) preparation of aseptic embryo mixed liquor: in step 2) sterile beaker in add sterilized water stirring, embryo is dispersed in sterilized water, dispersion is 90-100/mL, obtains aseptic embryo mixed liquor;
4) preparation of root media is sprouted: by culture medium based on 1/2MS culture medium, stereometer with this basal medium, following additive is added: naphthalene acetic acid (NAA) 0.9mg/L in this basal medium, activated carbon 0.4g/L, 6-benzyl aminoadenine (6-BA) 0.1mg/L, coconut juice 75g/L, peeling Fructus Musae 45g/L, rice 30g/L, sucrose 33g/L and agar 6.2g/L, obtain sprouting root media, and the pH value of this sprouting root media is adjusted to 6.2, afterwards the root media of sprouting of preparation is dispensed into in the 650mL tissue culture bottle of air-vent, the liquid filling degree of depth of each tissue culture bottle is 4cm, then by each tissue culture bottle in high-pressure sterilizing pot in 121 DEG C of sterilizing 23min, after cooling standby,
5) inoculation of embryo and cultivation: being inoculated into by aseptic embryo mixed liquor sterile pipette in sprouting root media, the inoculum concentration of each tissue culture bottle is 1000 �� L, then be coated with aseptic spreading rod and put down, by the bottle cap screwing of tissue culture bottle, inoculation completes; Afterwards by each tissue culture bottle dark treatment 5 days at 24 DEG C, then every day intensity of illumination be 1200-1500Lux, light application time be 10-11h culture environment under cultivate 35 days, last every day intensity of illumination be 2500-3000Lux, light application time be 12-13h culture environment under cultivate 115 days after, obtain candidum tissue culturing seedling, tissue cultured seedling is evenly distributed, Seedling body healthy and strong, transplanting survival rate is 93.5%, and aberration rate is zero.
In the step 3) of above example 1-3, following methods can be adopted to determine the dispersion of embryo: pipette the aseptic embryo mixed liquor of 1mL with liquid-transfering gun, after diluting 10 times with sterilized water, embryo is counted, determine its dispersion, if dispersion is unsatisfactory for the requirement of 60-100/mL, then in sterile beaker, supplement embryo or sterilized water, until dispersion meets requirement.
Claims (2)
1. a candidum tissue culturing inoculation method, it is characterised in that comprise the following steps:
1) selection of Herba Dendrobii fruit pod: the Herba Dendrobii selecting outdoor pseudo-wild cultivating 3-6 raw is tied and grown the maturation formed 110-130 days and do not ftracture really pod;
2) embryo sowing: by Herba Dendrobii fruit pod on superclean bench after the alcohol disinfecting 12-15min that mass percent concentration is 65-75%, then the embryo in fruit pod is seeded in sterile beaker;
3) preparation of aseptic embryo mixed liquor: in step 2) sterile beaker in add sterilized water stirring, embryo is dispersed in sterilized water, dispersion is 60-100/mL, obtains aseptic embryo mixed liquor;
4) preparation of root media is sprouted: by culture medium based on 1/2MS culture medium, stereometer with this basal medium, following additive is added: naphthalene acetic acid 0.7-0.9mg/L in this basal medium, activated carbon 0.2-0.5g/L, 6-benzyl aminoadenine 0.05-0.2mg/L, coconut juice 50-80g/L, peeling Fructus Musae 30-50g/L, rice 20-30g/L, sucrose 25-35g/L and agar 5.8-6.3g/L, obtain sprouting root media, and the pH value of this sprouting root media is adjusted to 6.0 ~ 6.2, afterwards the root media of sprouting of preparation is dispensed into in the 650mL tissue culture bottle of air-vent, the liquid filling degree of depth of each tissue culture bottle is 3-4cm, then by each tissue culture bottle in high-pressure sterilizing pot in 121 DEG C of sterilizing 20-23min, after cooling standby,
5) inoculation of embryo and cultivation: being inoculated into by aseptic embryo mixed liquor sterile pipette in sprouting root media, the inoculum concentration of each tissue culture bottle is 800-1000 �� L, then be coated with aseptic spreading rod and put down, by the bottle cap screwing of tissue culture bottle, inoculation completes; Afterwards by each tissue culture bottle dark treatment 5-7 days at 22-24 DEG C, then every day intensity of illumination be 1200-1500Lux, light application time be 10-11h culture environment under cultivate 30-40 days, last every day intensity of illumination be 2500-3000Lux, light application time be 12-13h culture environment under cultivate some skies, until embryo germination obtain candidum tissue culturing seedling.
2. a kind of candidum tissue culturing inoculation method according to claim 1, it is characterized in that in step 3), the defining method of the dispersion of embryo is: pipette the aseptic embryo mixed liquor of 1mL with liquid-transfering gun, after diluting 10 times with sterilized water, embryo is counted, it is determined that its dispersion, if dispersion is unsatisfactory for the requirement of 60-100/mL, in sterile beaker, then supplement embryo or sterilized water, until dispersion meets requirement.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109793010A (en) * | 2019-03-14 | 2019-05-24 | 华侨大学 | A kind of liquid compound bacterial agent for promoting the growth of Dendrobium officinale and using method thereof |
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| CN105104209A (en) * | 2015-09-24 | 2015-12-02 | 戴亚峰 | Method for cultivating tissues to obtain seedlings of dendrobium huoshanense at one step and formula of nutrient liquid for method |
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| CN109793010A (en) * | 2019-03-14 | 2019-05-24 | 华侨大学 | A kind of liquid compound bacterial agent for promoting the growth of Dendrobium officinale and using method thereof |
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