CN104938338A - Culture medium series for quick breeding and seedling of dendrobium chrysanthum and tissue culture method - Google Patents
Culture medium series for quick breeding and seedling of dendrobium chrysanthum and tissue culture method Download PDFInfo
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Abstract
The invention provides a culture medium series for quick breeding and seedling of dendrobium chrysanthum and a tissue culture method. The culture medium series comprises a sowing culture medium, a proliferation culture medium, a sound seedling culture medium, a differentiated seedling culture medium and a rooting culture medium. The tissue culture method comprises the following steps: at first, seeds are cultivated in a germination culture medium, and illuminated directly instead of being cultivated in the dark, after the seeds germinate to form protocorm, a callus is generated through induction in a protocorm proliferation and inducing culture medium, and the callus further forms a protocorm-like body; the protocorm like body is differentiated to form a small seedling; the small seedling is transferred to the sound seedling culture medium; after the small seedling grows into a large seedling, the large seedling is cultivated in the rooting culture medium to form a dendrobium officinale seedling. The culture medium series and the tissue culture method have the advantages that the cultivation time of dendrobium officinale can be shortened, not only is the operation simple, but also the limitation of time and places is eliminated, the annual production can be ensured through continuous differentiation and proliferation of the protocorm-like body, and the large-scale industrial production and application are facilitated.
Description
Technical field
The present invention relates to the propagation method of medicinal plant seedling, relate in particular to a kind of HERBA DENDROBII fast propagating culture medium series and tissue culture method.
Background technology
HERBA DENDROBII has another name called: golden hairpin stone, flat Jin Chai, flat yellow grass, flat grass are perennial herb, sending out hairpin and gaining the name on image head in ancient times.HERBA DENDROBII kind is careful as dust, and in a capsule, contained seed reaches 1,000,000 more than.Owing to lacking endosperm, seed could need be sprouted with mycosymbiosis, germination rate very low (less than 5%) under natural conditions.HERBA DENDROBII natural propagation power is extremely low, and Sterile culture is more difficult again, and for many years to the collection capacity of HERBA DENDROBII much larger than its amount of growth, wild HERBA DENDROBII natural propagation has been on the verge of disappearance.For development artificial cultivation is met the need of market, some appreciable varieties in medicinal dendrobium have become the emphasis of current tissue rapid propagation research.
Study on tissue culture at present about HERBA DENDROBII has relevant report, but the problem of the high cost needed for tissue cultures and test tube seedling cycle length is the bottleneck of the large production of restriction HERBA DENDROBII industrialization.Therefore need badly a kind of can the tissue culturing system of Fast-propagation HERBA DENDROBII, for promoting artificial culture technology and theory and practice basis is established in large-scale industrialized nursery.
Summary of the invention
For solving above-mentioned prior art Problems existing, the present invention starts with from HERBA DENDROBII seed culture, to the nutrient media components of HERBA DENDROBII tissue cultures, grope to find by experiment for many years, can Fast-propagation HERBA DENDROBII and can the tissue culturing system of whole year production, for factorial seedling growth provides technical support.This HERBA DENDROBII fast propagating culture medium series can significantly improve the sprout time of HERBA DENDROBII seed, directly 15-22 days is shortened to from the time of about traditional 75 days, and wild seed germination rate is up to 94.5-97.8%, HERBA DENDROBII plantlet in vitro transplanting survival rate is up to 98%.
The present invention proposes a kind of HERBA DENDROBII fast propagating culture medium series, and its technical scheme is achieved in that
A kind of medium series of HERBA DENDROBII Fast-propagation seedling, this medium series comprises sowing medium, proliferated culture medium, strong seedling culture base, differentiation seedling medium and root media, and each constituent content of described medium is respectively:
Sowing medium: 1000 milliliters of A liquid, 1000 milliliters of B liquid, 100 milliliters of C liquid, 100 milliliters of D liquid, 20 milliliters of L liquid, 4 milliliters of M1 liquid, 380-420g sucrose, 72-76g agar, 34-38g carbon dust, 1900-2100g potato, apple or bananas and 10 premium on currency;
Proliferated culture medium: 1000 milliliters of A liquid, 1000 milliliters of B liquid, 100 milliliters of C liquid, 100 milliliters of D liquid, 20 milliliters of L liquid, 8 milliliters of M1 liquid, 5 milliliters of M2 liquid, 380-420g sucrose, 72-76g agar, 34-38g carbon dust, 1900-2100g potato, apple or bananas and 10 premium on currency;
Strong seedling culture base: 1000 milliliters of A liquid, 1000 milliliters of B liquid, 100 milliliters of C liquid, 100 milliliters of D liquid, 20 milliliters of L liquid, 15 milliliters of M1 liquid, 10 milliliters of M2 liquid, 380-420g sucrose, 72-76g agar, 38-42g carbon dust, 1900-2100g potato, apple or bananas and 10 premium on currency;
Differentiation seedling medium: 1000 milliliters of A liquid, 1000 milliliters of B liquid, 100 milliliters of C liquid, 100 milliliters of D liquid, 20 milliliters of L liquid, 20 milliliters of M1 liquid, 10 milliliters of M2 liquid, 380-420g sucrose, 72-76g agar, 38-42g carbon dust, 1900-2100g potato, apple or bananas and 10 premium on currency;
Root media: 1000 milliliters of A liquid, 1000 milliliters of B liquid, 100 milliliters of C liquid, 100 milliliters of D liquid, 20 milliliters of L liquid, 20 milliliters of M1 liquid, 10 milliliters of M2 liquid, 480-520g sucrose, 74-78g agar, 38-42g carbon dust, 2400-2600g potato or bananas and 10 premium on currency;
Wherein, in described A liquid, B liquid, C liquid, D liquid, L liquid, M1 liquid and M2 liquid, Elemental Composition is not:
A liquid: 17.5g/L ammonium nitrate, 19.5g/L potassium nitrate, 4.3g/L magnesium sulfate and 2.4g/L potassium dihydrogen phosphate;
B liquid: 4.6g/L calcium chloride;
C liquid: 0.4g/L glycine, 0.05g/L vitamin B1,0.15g/L nicotinic acid, 0.1g/L hydrochloric acid ratio are trembled pungent VB6 and 40g inositol;
D liquid: 5.59g/L ferrous sulfate and 7.47g/L disodium ethylene diamine tetraacetate;
L liquid: 19.9g/L manganese sulphate, 9.2g/L zinc sulphate, 6.6g/L boric acid, 0.83g/L potassium iodide, 0.025g/L copper sulphate, 0.025g/L cobalt chloride and 0.25g/L sodium molybdate;
M1 liquid: 0.5g/La-naa and 0.44g/L sodium hydroxide;
M2 liquid: 0.55g/L 6-benzyladenine and 0.4g/L sodium hydroxide.
Further, the pH value of described sowing medium is 5.4-5.8, regulates the pH value of described sowing medium with acid-base modifier, and namely sodium hydroxide adjustment is then added in medium jaundice, and blackout is then added hydrochloric acid and regulated; The pH value of described proliferated culture medium is 5.4-5.8, regulates the pH value of described proliferated culture medium with acid-base modifier, and namely sodium hydroxide adjustment is then added in medium jaundice, and blackout is then added hydrochloric acid and regulated; The pH value of described strong seedling culture base is 5.4-5.8, regulates the pH value of described strong seedling culture base with acid-base modifier, and namely sodium hydroxide adjustment is then added in medium jaundice, and blackout is then added hydrochloric acid and regulated; The pH value of described differentiation seedling medium is 5.4-5.8, regulates the pH value of described differentiation seedling medium with acid-base modifier, and namely sodium hydroxide adjustment is then added in medium jaundice, and blackout is then added hydrochloric acid and regulated; The pH value of described seedling medium of taking root is 5.4-5.8, the pH value of seedling medium of taking root described in regulating with acid-base modifier, and namely sodium hydroxide adjustment is then added in medium jaundice, and blackout is then added hydrochloric acid and regulated.
Further, in described medium and described A liquid, B liquid, C liquid, D liquid, L liquid, M1 liquid and M2 liquid, water used is mineral water.
The invention also discloses the tissue culture method using above-mentioned medium series by HERBA DENDROBII Fast-propagation seedling, its step is as follows:
(1) the axenic germination of HERBA DENDROBII seed
Gather HERBA DENDROBII capsule seed, to carry out disinfection sterilizing with alcohol and liquor natrii hypochloritis successively, one end of seed after sterilizing is cut, Powdered embryo is sowed uniformly on sowing medium, 15-22 days is cultivated there being the group training room of illumination, the Seed Development protocorm sprouted, maintenance group training room temperature is at 22-25 DEG C;
(2) the propagation of HERBA DENDROBII protocorm
Evenly be transferred in proliferated culture medium by the protocorm that seed germination is formed, be placed in group training room and carry out illumination cultivation, maintenance group training room temperature is at 22-25 DEG C, and protocorm grows faint yellow callus, 22-27 days, and the continuous Isolation and proliferation of callus becomes protocorms;
(3) the differentiation of HERBA DENDROBII protocorms
Chosen by protocorms and be evenly transferred in differential medium, be placed in group training room and carry out illumination cultivation, maintenance group training room temperature is at 22-25 DEG C, 26-30 days, and protocorms is divided into seedling;
(4) the strong sprout of HERBA DENDROBII seedling
Be placed on by seedling on the large culture dish of sterilizing in advance, and seedling is inserted in strong seedling culture base, be placed in group training room and carry out illumination cultivation, maintenance group training room temperature is at 22-25 DEG C, 32-45 days, and seedling grows into the HERBA DENDROBII seedlings grown fine;
(5) the taking root of HERBA DENDROBII seedlings
Be placed on by seedlings on the large culture dish of sterilizing in advance, and seedlings are inserted in root media, be placed in group training room and carry out illumination cultivation, maintenance group training room temperature is at 22-25 DEG C, 32-60 days, and seedlings grow into complete HERBA DENDROBII plantlet in vitro.
Beneficial effect of the present invention:
The present invention, by regulating sowing nutrient media components, is made the HERBA DENDROBII seed sowed by this medium without the need to through the dark culturing time, directly carries out light treatment, the Seed Development protocorm sprouted for 15-22 days, and germination rate reaches 94.5-97.8%; Further, to the adjustment of formula of proliferated culture medium, strong seedling culture base, differential medium and root media, make the HERBA DENDROBII plantlet in vitro stalwartness grown out, transplanting survival rate is up to 98%.Root media preferably adopts banana, and the root media seedling time using banana is 32-45 days, and uses the root media seedling time of potato to be 46-60 days.
Accompanying drawing explanation
Fig. 1 is HERBA DENDROBII protocorm photo;
Fig. 2 is HERBA DENDROBII protocorms photo;
Fig. 3 is HERBA DENDROBII seedling photo;
Fig. 4 is HERBA DENDROBII plantlet in vitro photo.
Embodiment
Embodiment 1
Preparation A liquid: take ammonium nitrate 17.5g, potassium nitrate 19.5g, magnesium sulfate 4.3g, potassium dihydrogen phosphate 2.4g, then adds mixing in 1L water and dissolves.
Preparation B liquid: take calcium chloride 4.6g, then puts into the mixing of 1L water and dissolves.
Preparation C liquid: take glycine 0.4g, 0.05g vitamin B1, nicotinic acid 0.15g and the 0.1g hydrochloric acid ratio pungent VB6 that trembles adds and mix dissolving in 600ml water and obtain mixed liquor I, take 440g inositol 400ml water mixing dissolving again and obtain mixed liquor I I, mixed liquor I is mixed with mixed liquor I I.
Preparation D liquid: take 5.59g ferrous sulfate and 7.47g disodium ethylene diamine tetraacetate, after dissolving respectively with water, then mixing is mixed with 1L solution.
Preparation L liquid: take 19.9g/L manganese sulphate, 9.2g/L zinc sulphate, 6.6g/L boric acid, 0.83g/L potassium iodide, 0.025g/L copper sulphate and the water-soluble solution of 0.025g/L cobalt chloride, take the water-soluble solution of 0.25g sodium molybdate again, then two kinds of solution are mixed to add water be mixed with 1L solution.
Preparation M1 liquid: take 0.5g/La-naa and 0.44g/L sodium hydroxide, be mixed with solution with 1L water, put into Refrigerator store;
Preparation M2 liquid: take 0.55g/L 6-benzyladenine and 0.4g/L sodium hydroxide, be mixed with solution with 1L water, keep in Dark Place.
Preparation sowing medium: take 1000 milliliters of A liquid, 1000 milliliters of B liquid, 100 milliliters of C liquid, 100 milliliters of D liquid, 20 milliliters of L liquid and 4 milliliters of M1 liquid blendings, then 400g sucrose, 74g agar, 36g carbon dust and the potato of 2000g through pulverizing is added, add 10 premium on currency high-temperature heatings again, mix at 80 DEG C subsequently, bottling, carry out high-pressure sterilizing pot sterilizing, cool.
Preparation proliferated culture medium: take 1000 milliliters of A liquid, 1000 milliliters of B liquid, 100 milliliters of C liquid, 100 milliliters of D liquid, 20 milliliters of L liquid, 8 milliliters of M1 liquid and 5 milliliters of M2 liquid blendings, then 400g sucrose, 74g agar, 36g carbon dust and the potato of 2000g through pulverizing is added, add 10 premium on currency high-temperature heatings again, mix at 80 DEG C subsequently, bottling, carry out high-pressure sterilizing pot sterilizing, cool.
Preparation strong seedling culture base: taking 1000 milliliters of A liquid, 1000 milliliters of B liquid, 100 milliliters of C liquid, 100 milliliters of D liquid, 20 milliliters of L liquid, 15 milliliters of M1 liquid and 10 milliliters of M2 liquid blendings, then adding 400g sucrose, 74g agar, 40g carbon dust and the potato of 2000g through pulverizing, add 10 premium on currency high-temperature heatings again, mix at 80 DEG C subsequently, bottling, carry out high-pressure sterilizing pot sterilizing, cool.
Preparation differentiation seedling medium: take 1000 milliliters of A liquid, 1000 milliliters of B liquid, 100 milliliters of C liquid, 100 milliliters of D liquid, 20 milliliters of L liquid, 20 milliliters of M1 liquid and 10 milliliters of M2 liquid blendings, then 400g sucrose, 74g agar, 40g carbon dust and the potato of 2000g through pulverizing is added, add 10 premium on currency high-temperature heatings again, mix at 80 DEG C subsequently, bottling, carry out high-pressure sterilizing pot sterilizing, cool.
Preparation root media: take 1000 milliliters of A liquid, 1000 milliliters of B liquid, 100 milliliters of C liquid, 100 milliliters of D liquid, 20 milliliters of L liquid, 20 milliliters of M1 liquid and 10 milliliters of M2 liquid blendings, then 500g sucrose, 76g agar, 40g carbon dust and the banana of 2500g through pulverizing is added, add 10 premium on currency high-temperature heatings again, mix at 80 DEG C subsequently, bottling, carry out high-pressure sterilizing pot sterilizing, cool.
The tissue culture method of HERBA DENDROBII Fast-propagation seedling, its step is as follows:
(1) the axenic germination of HERBA DENDROBII seed
Gather HERBA DENDROBII capsule seed, to carry out disinfection sterilizing with alcohol and liquor natrii hypochloritis successively, one end of seed after sterilizing is cut, Powdered embryo is sowed uniformly on sowing medium, train room there being the group of illumination and cultivate 15 days, the seed sprouted just forms protocorm (see Fig. 1), and maintenance group training room temperature is at 22-25 DEG C; Calculate according to bottle number, germination rate reaches 97.8%.
(2) the propagation of HERBA DENDROBII protocorm
The protocorm that seed germination is formed evenly is transferred in proliferated culture medium, be placed in group training room and carry out illumination cultivation, maintenance group training room temperature is at 22-25 DEG C, and protocorm grows faint yellow callus, 22 days, the continuous Isolation and proliferation of callus became protocorms (see Fig. 2);
(3) the differentiation of HERBA DENDROBII protocorms
Chosen by protocorms and be evenly transferred in differential medium, be placed in group training room and carry out illumination cultivation, maintenance group training room temperature is at 22-25 DEG C, and 26 days, protocorms was divided into seedling (see Fig. 3);
(4) the strong sprout of HERBA DENDROBII seedling
Be placed on by seedling on the large culture dish of sterilizing in advance, and seedling is inserted in strong seedling culture base, be placed in group training room and carry out illumination cultivation, maintenance group training room temperature is at 22-25 DEG C, and 32 days, seedling grew into the HERBA DENDROBII seedlings grown fine;
(5) the taking root of HERBA DENDROBII seedlings
Seedlings are placed on the large culture dish of sterilizing in advance, and seedlings are inserted in root media, be placed in group training room and carry out illumination cultivation, maintenance group training room temperature is at 22-25 DEG C, 32 days, seedlings grew into complete HERBA DENDROBII plantlet in vitro (see Fig. 4).
HERBA DENDROBII plantlet in vitro is taken out bottle seedling after natural daylight lower refining seedling one week and cleans root medium, transplant to peat soil and liver moss volume ratio be that in the mixed-matrix of 1:1, survival rate is more than 98%.
Embodiment 2
A liquid, B liquid, C liquid, D liquid, L liquid, M1 liquid are identical with embodiment 1 with compound method with the content of M2 liquid.
Preparation sowing medium: take 1000 milliliters of A liquid, 1000 milliliters of B liquid, 100 milliliters of C liquid, 100 milliliters of D liquid, 20 milliliters of L liquid and 8 milliliters of M1 liquid blendings, then 380g sucrose, 72g agar, 34g carbon dust and the banana of 1900g through pulverizing is added, add 10 premium on currency high-temperature heatings again, mix at 80 DEG C subsequently, bottling, carry out high-pressure sterilizing pot sterilizing, cool.
Preparation proliferated culture medium: take 1000 milliliters of A liquid, 1000 milliliters of B liquid, 100 milliliters of C liquid, 100 milliliters of D liquid, 20 milliliters of L liquid, 8 milliliters of M1 liquid and 5 milliliters of M2 liquid blendings, then 380g sucrose, 72g agar, 34g carbon dust and the banana of 1900g through pulverizing is added, add 10 premium on currency high-temperature heatings again, mix at 80 DEG C subsequently, bottling, carry out high-pressure sterilizing pot sterilizing, cool.
Preparation strong seedling culture base: taking 1000 milliliters of A liquid, 1000 milliliters of B liquid, 100 milliliters of C liquid, 100 milliliters of D liquid, 20 milliliters of L liquid, 15 milliliters of M1 liquid and 10 milliliters of M2 liquid blendings, then adding 380g sucrose, 72g agar, 38g carbon dust and the banana of 1900g through pulverizing, add 10 premium on currency high-temperature heatings again, mix at 80 DEG C subsequently, bottling, carry out high-pressure sterilizing pot sterilizing, cool.
Preparation differentiation seedling medium: take 1000 milliliters of A liquid, 1000 milliliters of B liquid, 100 milliliters of C liquid, 100 milliliters of D liquid, 20 milliliters of L liquid, 20 milliliters of M1 liquid and 10 milliliters of M2 liquid blendings, then 380g sucrose, 72g agar, 38g carbon dust and the banana of 1900g through pulverizing is added, add 10 premium on currency high-temperature heatings again, mix at 80 DEG C subsequently, bottling, carry out high-pressure sterilizing pot sterilizing, cool.
Preparation root media: take 1000 milliliters of A liquid, 1000 milliliters of B liquid, 100 milliliters of C liquid, 100 milliliters of D liquid, 20 milliliters of L liquid, 20 milliliters of M1 liquid and 10 milliliters of M2 liquid blendings, then 480g sucrose, 74g agar, 38g carbon dust and the banana of 2400g through pulverizing is added, add 10 premium on currency high-temperature heatings again, mix at 80 DEG C subsequently, bottling, carry out high-pressure sterilizing pot sterilizing, cool.
The tissue culture method of HERBA DENDROBII Fast-propagation seedling, its step is as follows:
(1) the axenic germination of HERBA DENDROBII seed
Gather HERBA DENDROBII capsule seed, to carry out disinfection sterilizing with alcohol and liquor natrii hypochloritis successively, one end of seed after sterilizing is cut, Powdered embryo is sowed uniformly on sowing medium, train room there being the group of illumination and cultivate 22 days, the seed sprouted just forms protocorm, and maintenance group training room temperature is at 22-25 DEG C; Calculate according to bottle number, germination rate reaches 94.5%.
(2) the propagation of HERBA DENDROBII protocorm
Evenly be transferred in proliferated culture medium by the protocorm that seed germination is formed, be placed in group training room and carry out illumination cultivation, maintenance group training room temperature is at 22-25 DEG C, and protocorm grows faint yellow callus, 27 days, and the continuous Isolation and proliferation of callus becomes protocorms;
(3) the differentiation of HERBA DENDROBII protocorms
Chosen by protocorms and be evenly transferred in differential medium, be placed in group training room and carry out illumination cultivation, maintenance group training room temperature is at 22-25 DEG C, and 30 days, protocorms was divided into seedling;
(4) the strong sprout of HERBA DENDROBII seedling
Be placed on by seedling on the large culture dish of sterilizing in advance, and seedling is inserted in strong seedling culture base, be placed in group training room and carry out illumination cultivation, maintenance group training room temperature is at 22-25 DEG C, and 45 days, seedling grew into the HERBA DENDROBII seedlings grown fine;
(5) the taking root of HERBA DENDROBII seedlings
Be placed on by seedlings on the large culture dish of sterilizing in advance, and seedlings are inserted in root media, be placed in group training room and carry out illumination cultivation, maintenance group training room temperature is at 22-25 DEG C, and 45 days, seedlings grew into complete HERBA DENDROBII plantlet in vitro.
HERBA DENDROBII plantlet in vitro is taken out bottle seedling after natural daylight lower refining seedling one week and cleans root medium, transplant to peat soil and liver moss volume ratio be that in the mixed-matrix of 1:1, survival rate is more than 98%.
Embodiment 3
A liquid, B liquid, C liquid, D liquid, L liquid, M1 liquid are identical with embodiment 1 with compound method with the content of M2 liquid.
Preparation sowing medium: take 1000 milliliters of A liquid, 1000 milliliters of B liquid, 100 milliliters of C liquid, 100 milliliters of D liquid, 20 milliliters of L liquid and 8 milliliters of M1 liquid blendings, then 420g sucrose, 76g agar, 38g carbon dust and the apple of 2100g through pulverizing is added, add 10 premium on currency high-temperature heatings again, mix at 80 DEG C subsequently, bottling, carry out high-pressure sterilizing pot sterilizing, cool.
Preparation proliferated culture medium: take 1000 milliliters of A liquid, 1000 milliliters of B liquid, 100 milliliters of C liquid, 100 milliliters of D liquid, 20 milliliters of L liquid, 8 milliliters of M1 liquid and 5 milliliters of M2 liquid blendings, then 420g sucrose, 76g agar, 38g carbon dust and the apple of 2100g through pulverizing is added, add 10 premium on currency high-temperature heatings again, mix at 80 DEG C subsequently, bottling, carry out high-pressure sterilizing pot sterilizing, cool.
Preparation strong seedling culture base: taking 1000 milliliters of A liquid, 1000 milliliters of B liquid, 100 milliliters of C liquid, 100 milliliters of D liquid, 20 milliliters of L liquid, 15 milliliters of M1 liquid and 10 milliliters of M2 liquid blendings, then adding 420g sucrose, 76g agar, 42g carbon dust and the apple of 2100g through pulverizing, add 10 premium on currency high-temperature heatings again, mix at 80 DEG C subsequently, bottling, carry out high-pressure sterilizing pot sterilizing, cool.
Preparation differentiation seedling medium: take 1000 milliliters of A liquid, 1000 milliliters of B liquid, 100 milliliters of C liquid, 100 milliliters of D liquid, 20 milliliters of L liquid, 20 milliliters of M1 liquid and 10 milliliters of M2 liquid blendings, then 420g sucrose, 76g agar, 42g carbon dust and the apple of 2100g through pulverizing is added, add 10 premium on currency high-temperature heatings again, mix at 80 DEG C subsequently, bottling, carry out high-pressure sterilizing pot sterilizing, cool.
Preparation root media: take 1000 milliliters of A liquid, 1000 milliliters of B liquid, 100 milliliters of C liquid, 100 milliliters of D liquid, 20 milliliters of L liquid, 20 milliliters of M1 liquid and 10 milliliters of M2 liquid blendings, then 520g sucrose, 78g agar, 42g carbon dust and the potato of 2600g through pulverizing is added, add 10 premium on currency high-temperature heatings again, mix at 80 DEG C subsequently, bottling, carry out high-pressure sterilizing pot sterilizing, cool.
The tissue culture method of HERBA DENDROBII Fast-propagation seedling, its step is as follows:
(1) the axenic germination of HERBA DENDROBII seed
Gather HERBA DENDROBII capsule seed, to carry out disinfection sterilizing with alcohol and liquor natrii hypochloritis successively, one end of seed after sterilizing is cut, Powdered embryo is sowed uniformly on sowing medium, train room there being the group of illumination and cultivate 19 days, the seed sprouted just forms protocorm, and maintenance group training room temperature is at 22-25 DEG C; Calculate according to bottle number, germination rate reaches 97%.
(2) the propagation of HERBA DENDROBII protocorm
Evenly be transferred in proliferated culture medium by the protocorm that seed germination is formed, be placed in group training room and carry out illumination cultivation, maintenance group training room temperature is at 22-25 DEG C, and protocorm grows faint yellow callus, 25 days, and the continuous Isolation and proliferation of callus becomes protocorms;
(3) the differentiation of HERBA DENDROBII protocorms
Chosen by protocorms and be evenly transferred in differential medium, be placed in group training room and carry out illumination cultivation, maintenance group training room temperature is at 22-25 DEG C, and 28 days, protocorms was divided into seedling;
(4) the strong sprout of HERBA DENDROBII seedling
Be placed on by seedling on the large culture dish of sterilizing in advance, and seedling is inserted in strong seedling culture base, be placed in group training room and carry out illumination cultivation, maintenance group training room temperature is at 22-25 DEG C, and 40 days, seedling grew into the HERBA DENDROBII seedlings grown fine;
(5) the taking root of HERBA DENDROBII seedlings
Be placed on by seedlings on the large culture dish of sterilizing in advance, and seedlings are inserted in root media, be placed in group training room and carry out illumination cultivation, maintenance group training room temperature is at 22-25 DEG C, and 60 days, seedlings grew into complete HERBA DENDROBII plantlet in vitro.
HERBA DENDROBII plantlet in vitro is taken out bottle seedling after natural daylight lower refining seedling one week and cleans root medium, transplant to peat soil and liver moss volume ratio be that in the mixed-matrix of 1:1, survival rate is more than 98%.
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, within the spirit and principles in the present invention all, any amendment done, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (3)
1. the medium series of a HERBA DENDROBII Fast-propagation seedling, it is characterized in that, this medium series comprises sowing medium, proliferated culture medium, strong seedling culture base, differentiation seedling medium and root media, and each constituent content of described medium is respectively:
Sowing medium: 1000 milliliters of A liquid, 1000 milliliters of B liquid, 100 milliliters of C liquid, 100 milliliters of D liquid, 20 milliliters of L liquid, 4 milliliters of M1 liquid, 380-420g sucrose, 72-76g agar, 34-38g carbon dust, 1900-2100g potato, apple or bananas and 10 premium on currency;
Proliferated culture medium: 1000 milliliters of A liquid, 1000 milliliters of B liquid, 100 milliliters of C liquid, 100 milliliters of D liquid, 20 milliliters of L liquid, 8 milliliters of M1 liquid, 5 milliliters of M2 liquid, 380-420g sucrose, 72-76g agar, 34-38g carbon dust, 1900-2100g potato, apple or bananas and 10 premium on currency;
Strong seedling culture base: 1000 milliliters of A liquid, 1000 milliliters of B liquid, 100 milliliters of C liquid, 100 milliliters of D liquid, 20 milliliters of L liquid, 15 milliliters of M1 liquid, 10 milliliters of M2 liquid, 380-420g sucrose, 72-76g agar, 38-42g carbon dust, 1900-2100g potato, apple or bananas and 10 premium on currency;
Differentiation seedling medium: 1000 milliliters of A liquid, 1000 milliliters of B liquid, 100 milliliters of C liquid, 100 milliliters of D liquid, 20 milliliters of L liquid, 20 milliliters of M1 liquid, 10 milliliters of M2 liquid, 380-420g sucrose, 72-76g agar, 38-42g carbon dust, 1900-2100g potato, apple or bananas and 10 premium on currency;
Root media: 1000 milliliters of A liquid, 1000 milliliters of B liquid, 100 milliliters of C liquid, 100 milliliters of D liquid, 20 milliliters of L liquid, 20 milliliters of M1 liquid, 10 milliliters of M2 liquid, 480-520g sucrose, 74-78g agar, 38-42g carbon dust, 2400-2600g potato or bananas and 10 premium on currency;
Wherein, in described A liquid, B liquid, C liquid, D liquid, L liquid, M1 liquid and M2 liquid, Elemental Composition is not:
A liquid: 17.5g/L ammonium nitrate, 19.5g/L potassium nitrate, 4.3g/L magnesium sulfate and 2.4g/L potassium dihydrogen phosphate;
B liquid: 4.6g/L calcium chloride;
C liquid: 0.4g/L glycine, 0.05g/L vitamin B1,0.15g/L nicotinic acid, 0.1g/L hydrochloric acid ratio are trembled pungent VB6 and 40g inositol;
D liquid: 5.59g/L ferrous sulfate and 7.47g/L disodium ethylene diamine tetraacetate;
L liquid: 19.9g/L manganese sulphate, 9.2g/L zinc sulphate, 6.6g/L boric acid, 0.83g/L potassium iodide, 0.025g/L copper sulphate, 0.025g/L cobalt chloride and 0.25g/L sodium molybdate;
M1 liquid: 0.5g/La-naa and 0.44g/L sodium hydroxide;
M2 liquid: 0.55g/L 6-benzyladenine and 0.4g/L sodium hydroxide.
2., according to the medium series of claim 1 HERBA DENDROBII Fast-propagation seedling, it is characterized in that, the pH value of described sowing medium is 5.4-5.8, regulates the pH value of described sowing medium with acid-base modifier; The pH value of described proliferated culture medium is 5.4-5.8, regulates the pH value of described proliferated culture medium with acid-base modifier; The pH value of described strong seedling culture base is 5.4-5.8, regulates the pH value of described strong seedling culture base with acid-base modifier; The pH value of described differentiation seedling medium is 5.4-5.8, regulates the pH value of described differentiation seedling medium with acid-base modifier; The pH value of described seedling medium of taking root is 5.4-5.8, the pH value of seedling medium of taking root described in regulating with acid-base modifier.
3. use the series of medium described in claim 1 by a tissue culture method for HERBA DENDROBII Fast-propagation seedling, its feature comprises the following steps:
(1) the axenic germination of HERBA DENDROBII seed
Gather HERBA DENDROBII capsule seed, to carry out disinfection sterilizing with alcohol and liquor natrii hypochloritis successively, one end of seed after sterilizing is cut, Powdered embryo is sowed uniformly on sowing medium, 14-25 days is cultivated there being the group training room of illumination, the Seed Development protocorm sprouted, maintenance group training room temperature is at 22-25 DEG C;
(2) the propagation of HERBA DENDROBII protocorm
Evenly be transferred in proliferated culture medium by the protocorm that seed germination is formed, be placed in group training room and carry out illumination cultivation, maintenance group training room temperature is at 22-25 DEG C, and protocorm grows faint yellow callus, 22-27 days, and the continuous Isolation and proliferation of callus becomes protocorms;
(3) the differentiation of HERBA DENDROBII protocorms
Chosen by protocorms and be evenly transferred in differential medium, be placed in group training room and carry out illumination cultivation, maintenance group training room temperature is at 22-25 DEG C, 26-30 days, and protocorms is divided into seedling;
(4) the strong sprout of HERBA DENDROBII seedling
Be placed on by seedling on the large culture dish of sterilizing in advance, and seedling is inserted in strong seedling culture base, be placed in group training room and carry out illumination cultivation, maintenance group training room temperature is at 22-25 DEG C, 32-45 days, and seedling grows into the HERBA DENDROBII seedlings grown fine;
(5) the taking root of HERBA DENDROBII seedlings
Be placed on by seedlings on the large culture dish of sterilizing in advance, and seedlings are inserted in root media, be placed in group training room and carry out illumination cultivation, maintenance group training room temperature is at 22-25 DEG C, 32-60 days, and seedlings grow into complete HERBA DENDROBII plantlet in vitro.
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