CN104429973A - Method of culturing dendrobium officinale plantlets - Google Patents

Method of culturing dendrobium officinale plantlets Download PDF

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Publication number
CN104429973A
CN104429973A CN201410828043.9A CN201410828043A CN104429973A CN 104429973 A CN104429973 A CN 104429973A CN 201410828043 A CN201410828043 A CN 201410828043A CN 104429973 A CN104429973 A CN 104429973A
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medium
culturing
candidum
proliferation
subculture
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郑毓华
于太和
周道阳
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ANHUI TONGCHUANG MODERN AGRICULTURAL INVESTMENT DEVELOPMENT Co Ltd
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ANHUI TONGCHUANG MODERN AGRICULTURAL INVESTMENT DEVELOPMENT Co Ltd
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Abstract

The invention discloses a method of culturing dendrobium officinale plantlets and belongs to the field of plant tissue culturing technology. The method comprises the steps: step 1, induction and culturing of seeds: choosing dendrobium officinale fruit pods as explants, spreading seeds in the fruit pods on an explant induction culture medium and inducing seeds to germinate to form sprouts; step 2, multiplication and subculture of sprouts: grafting the sprouts on a multiplication and subculture medium, placing the multiplication and subculture medium in a culturing room and culturing to obtain dendrobium officinale seedlings; step 3, culturing of rooting seedlings: grafting the dendrobium officinale seedlings on a rooting culture medium and culturing to obtain strong dendrobium officinale seedlings; and step 4, transplanting: hardening the strong dendrobium officinale seedlings subjected to the rooting culturing and then transplanting the strong seedlings. The method of culturing dendrobium officinale plantlets solves problems of slow rooting and lower survival rate in the traditional culturing method of dendrobium officinale and provides convenience to the large-scale culturing of high-quality dendrobium officinale plantlets.

Description

A kind of cultural method of candidum tissue culturing seedling
Technical field
The invention belongs to field of plant tissue culture technique, more particularly, relate to a kind of cultural method of candidum tissue culturing seedling.
Background technology
Dendrobium candidum is perennial herb, belong to epiphytic orchid class, popular name Tiepi Fengdou, because its old stem is outward in black, have another name called for ribbed hedyotis herb, the stem of noble dendrobium is conventional rare Chinese medicine, and applicating history is long, be listed in top grade in Shennong's Herbal, now confirmed that it has nourishing Yin and clearing heat, the beneficial stomach that promotes the production of body fluid, relieving cough and moistening lung, the improving eyesight that wets one's whistle, improves immune function of human body, anti-curing oncoma and angiocardiopathy and improving water flood, prevents and treats cataract, the effect such as to promote longevity.But because growing environment is special and distribution limitation, and excavate for a long time, natural resources is increasingly exhausted, is classified as the natural crude drugs of focused protection by country and encourages artificial commerial growing dendrobium candidum.
Plant Tissue Breeding has been widely used in the every field of production practices as a kind of effective technological means.The cultivation of traditional dendrobium candidum is mainly by the mode of cuttage, plant division, but its reproduction rate is lower, and take root slowly, growth cycle is long.In order to meet the demand in market, domestic and international many scientific research personnel have carried out the research of dendrobium candidum tissue culture technique, and expect carry out producing in batches and improve reproduction coefficient and the transplanting survival rate of seedling, this is current problem demanding prompt solution.And the transplanting survival rate of seedling will be improved, the culture medium prescription for tissue cultures is most important.
By patent retrieval, about the technical scheme of dendrobium candidum tissue culture technique, existing related art scheme is open.As Chinese Patent Application No. 201310173395.0, the applying date is: 2013.05.13, invention and created name is: a kind of candidum tissue culturing seedling method, this application case discloses a kind of candidum tissue culturing seedling method, comprise following operating procedure: the medium making candidum tissue culturing seedling, load in container by following masses proportioning, then boiled, through liquid filling machine, medium is poured in tissue culture bottle, sterilizing, 1000 ~ 1500 grams of bananas stir into pasty state, white sugar 200 ~ 300 grams, 90 ~ 105 milliliters, ferrous sulfate, 25 ~ 33 grams, ammonium nitrate, 35 ~ 40 grams, potassium nitrate, 5 ~ 8 grams, magnesium sulfate, glycine 8 ~ 12 milliliters, VB11 ~ 3 milliliter, the each milliliter in B21 ~ 3, glycoprotein amino acid 20 ~ 30 milliliters, methyl α-naphthyl acetate 20 ~ 30 milliliters, 1000 milliliters, running water, sow on the above medium with the pollen of ripe Seeds of Dendrobium Candidum, grow through 3 months stepmother's seedlings.
And for example, Chinese Patent Application No. 201210102034.2, the applying date is: 2012.03.31, invention and created name is: a kind of dendrobium candidum is without the method for hormone tissue cultures, this application case discloses the method for a kind of dendrobium candidum without hormone tissue cultures, the method comprises Seeds of Dendrobium Candidum and sprouts for ball stem, the growth of ball stem, Proliferation, Differentiation, taking root of stem section, four tissue cultures stages of strong sprout, this four-stage medium used is followed successively by: without the MS medium of hormone, without MS minimal medium+mashed potatoes 15 ~ 20g/L medium of hormone, MS minimal medium+banana puree 15 ~ 20g/L+ mashed potatoes 15 ~ 20g/L medium without hormone and the 1/2MS minimal medium without hormone+banana puree 15 ~ 20g/L+ mashed potatoes 15 ~ 20g/L medium.In above-mentioned two application cases: technical scheme disclosed in Chinese Patent Application No. 201310173395.0 sows on the above medium with the pollen of ripe Seeds of Dendrobium Candidum, grow through 3 months stepmother's seedlings, then artificial infection divides seedling, this training method cannot ensure survival rate in actual applications, applies difficulty large.Disclosed in Chinese Patent Application No. 201210102034.2, technical scheme mainly have employed without hormone tissue culture technique, but the formula of its medium is unreasonable, requires further improvement.
Summary of the invention
1. invent the technical problem that will solve
The object of the invention is to overcome the deficiency of candidum tissue culturing seedling culture technique in prior art, provide a kind of cultural method of candidum tissue culturing seedling, adopt technical scheme of the present invention, reproduction coefficient and the transplanting survival rate of candidum tissue culturing seedling can be improved.
2. technical scheme
For achieving the above object, technical scheme provided by the invention is:
The cultural method of a kind of candidum tissue culturing seedling of the present invention, the steps include:
The bringing out and cultivate of step one, seed
Choose dendrobium candidum fruit pod as explant, dendrobium candidum fruit pod is cut with aseptic cutter, seed in fruit pod is spread out on explant induction medium, induction seed germination, be placed on culturing rack by explant induction medium, cultivation temperature is 23 ~ 25 DEG C, and light application time is 12 hours/day, cultivate 30 ~ 45 days under intensity of illumination 1500Lx condition, seed germination forms plumule;
Step 2, plumule proliferation and subculture are cultivated
The plumule formed in step one is transferred and to cultivate on proliferation and subculture medium, be placed in culturing room by proliferation and subculture medium, cultivation temperature is 23 ~ 25 DEG C, and light application time is 12 hours/day, cultivate 80 ~ 90 days under intensity of illumination 1800 ~ 2000Lx condition, obtain dendrobium candidum seedling;
The cultivation of step 3, seedling of taking root
Be transferred on root media by the dendrobium candidum seedling obtained in step 2, cultivate it, cultivation temperature is 25 ~ 26 DEG C, and light application time is 12 hours/day, cultivates 100 ~ 120 days under intensity of illumination 1800 ~ 2000Lx condition, obtains dendrobium candidum strong sprout;
Step 4, transplanting
Transplant after dendrobium candidum after step 3 is carried out culture of rootage carries out hardening strong sprout.
Further, explant induction medium in step one, using MS medium as minimal medium, adds agar 5 ~ 9g/L in minimal medium, white sugar 28 ~ 30g/L, active carbon 0.5 ~ 0.6g/L, banana 65 ~ 75g/L, self-control organic fertilizer 1.5 ~ 2g/L, bactericidal agent 1.5 ~ 2.5ml/L, the pH of explant induction medium is 5.4 ~ 5.8, carry out disinfecting action to above-mentioned explant induction medium, its sterilising temp is 120 ~ 126 DEG C, and sterilization time is 10 ~ 40min.
Further, proliferation and subculture medium in step 2 is using MS medium as minimal medium, agar 5 ~ 9g/L is added, white sugar 30 ~ 32g/L, active carbon 0.48 ~ 0.5g/L in minimal medium, banana 45 ~ 50g/L, bactericidal agent 1.5 ~ 2.5ml/L, the pH of proliferation and subculture medium is 5.4 ~ 5.8, carries out disinfecting action to above-mentioned proliferation and subculture medium, its sterilising temp is 120 ~ 126 DEG C, and sterilization time is 10 ~ 40min.
Further, root media in step 3, to make mother liquor by oneself as minimal medium, adds agar 5 ~ 9g/L in minimal medium, white sugar 20 ~ 22g/L, active carbon 0.5 ~ 0.55g/L, banana 50 ~ 55g/L, self-control natural organic liquid fertilizer 1.5 ~ 1.8g/L, bactericidal agent 1.5 ~ 2.5ml/L, the pH of root media is 5.4 ~ 5.8, carry out disinfecting action to above-mentioned root media, its sterilising temp is 120 ~ 126 DEG C, and sterilization time is 10 ~ 40min.
Further, described explant induction medium is identical with the self-control natural organic liquid fertilizer formula added in root media, and the formula of this self-control natural organic liquid fertilizer is specific as follows: raw bran 3000g, lactic acid bacteria 500g, whole milk powder 500g, water 50000g, rice bran 3000g, saccharomycete 500g, phosphate fertilizer 500g, brown sugar 6000g, sea-tangle 3000g, streaky pork 500g, fishbone dust 500g, 6, egg.
Further, the formula of the self-control mother liquor in described root media is specially: ammonium sulfate 0.46g/L, calcium chloride 0.17g/L, inositol 0.1g/L, potassium nitrate 2.83g/L, ferrous sulfate 0.03g/L, 1-methyl α-naphthyl acetate 2mg/L, potassium dihydrogen phosphate 0.4g/L, magnesium sulfate 0.19g/L.
3. beneficial effect
Adopt technical scheme provided by the invention, compared with prior art, there is following remarkable result:
(1) cultural method of a kind of candidum tissue culturing seedling of the present invention, specific natural organic matter consumption and medium is adopted to cultivate it by cultivating each stage at dendrobium candidum tissue, what the cultural method solving traditional dendrobium candidum brought takes root slowly, the problem that survival rate is not high, the seedling strain rugosity that the medium that the candidum tissue culturing seedling cultivated does not add natural organic matter is cultivated, highly, the number of blade all has to be increased and has good facilitation to root system, the plantlet in vitro of dendrobium candidum is bred be effectively improved, and then promote that the industrialization of dendrobium candidum is produced.
(2) cultural method of a kind of candidum tissue culturing seedling of the present invention, it is easy and simple to handle, with short production cycle, plant shoot survival percent high, medium configuration is convenient, nutritious, and condition of culture is simple, being convenient to management, providing convenient for cultivating high-quality dendrobium candidum seedling on a large scale.
Embodiment
Below in conjunction with embodiment, the present invention is described in detail, but they are not to further restriction of the present invention.
Embodiment 1
A kind of candidum tissue culturing seedling medium of the present embodiment, this medium comprises explant induction medium, proliferation and subculture medium and root media, wherein: described explant induction medium is for disseminating the seed in dendrobium candidum fruit pod, and induction seed germination forms plumule; Described plumule proliferation and subculture is cultivated for transferring plumule cultivating, and obtains dendrobium candidum seedling; Described root media is used for proceeding to dendrobium candidum seedling and cultivating, and obtains dendrobium candidum strong sprout.
Specifically in the present embodiment, explant induction medium, proliferation and subculture medium and root media are specific as follows: explant induction medium is using MS medium as minimal medium, agar 5.5g/L is added in minimal medium, white sugar 30g/L, active carbon 0.5g/L, banana 67g/L, self-control organic fertilizer 2g/L, bactericidal agent 2.5ml/L.In the present embodiment, the preparation process of explant inducing culture is specific as follows: (1), take medicine according to the composition of MS medium and consumption, makes it fully dissolve, and takes successively by respective concentration.(2), in MS medium, all kinds of material is added by required, it is respectively: agar 5.5g/L, white sugar 30g/L, active carbon 0.5g/L, banana 67g/L, self-control organic fertilizer 2g/L, bactericidal agent 2.5ml/L, wherein: the banana in the present embodiment refers to banana puree, this banana puree is removed the peel by banana, and being put in after weighing in cooking machine adds water breaks into banana puree; The detailed process adding all kinds of material in MS medium is: distilled water heating is boiled, after getting distilled water dissolving white sugar, add agar and stir, treat that it melts completely, active carbon, banana puree, self-control organic fertilizer, bactericidal agent, minimal medium are added simultaneously, survey pH value with pH test paper, and be 5.8 by NaOH or the HCI adjust ph of 0.1mol/L, finally adding water, it is volume required to be settled to.(3), by filling in blake bottle for the explant induction medium prepared, then seal with bottle cap, put into high-pressure steam sterilizing pan sterilizing, its sterilising temp is 120 ~ 126 DEG C, sterilization time is 10 ~ 40min, sterilized good medium is placed in condensation chamber, observe pollution-free after namely can be used for inoculation.Plant growth regulator is not added in explant induction medium in the present embodiment, object is exactly to improve the environment of biological cells and tissues reproduction restraint, explant induction medium in the present embodiment adopts natural banana puree and specific self-control organic fertilizer can promote the reproduction restraint of biological cells and tissues, safe and environmentally friendly.As technological improvement crucial in the present invention, the trace element made by oneself in the present embodiment in organic fertilizer is various, can be more balanced be beneficial to plant growth, for the reproduction coefficient and transplanting survival rate improving candidum tissue culturing seedling provides guarantee.In addition, white sugar provides substrate and energy for cell respiration metabolism, it than glucose low price many, cost-saving.
Proliferation and subculture medium in the present embodiment, using MS medium as minimal medium, adds agar 6g/L in minimal medium, white sugar 30g/L, active carbon 0.5g/L, banana 50g/L, bactericidal agent 2.5ml/L.In the present embodiment, the preparation process of proliferation and subculture medium is specific as follows: (1), take medicine according to the composition of MS medium and consumption, makes it fully dissolve, and takes successively by respective concentration.(2), in MS medium, all kinds of material is added by required, it is respectively: agar 6g/L, white sugar 30g/L, active carbon 0.5g/L, banana 50g/L, bactericidal agent 2.5ml/L, wherein: the banana in the present embodiment refers to banana puree, this banana puree is removed the peel by banana, and being put in after weighing in cooking machine adds water breaks into banana puree; The detailed process adding all kinds of material in MS medium is: distilled water heating is boiled, after getting distilled water dissolving white sugar, add agar and stir, treat that it melts completely, active carbon, banana puree, bactericidal agent, minimal medium are added simultaneously, survey pH value with pH test paper, and be 5.8 by NaOH or the HCI adjust ph of 0.1mol/L, finally adding water, it is volume required to be settled to.(3), by filling in blake bottle for the proliferation and subculture medium prepared, then seal with bottle cap, put into high-pressure steam sterilizing pan sterilizing, its sterilising temp is 120 ~ 126 DEG C, sterilization time is 10 ~ 40min, sterilized good medium is placed in condensation chamber, observe pollution-free after namely can be used for inoculation.Proliferation and subculture medium in the present embodiment does not add plant growth regulator, and by natural banana puree as nutrient component, adopting the specific proliferation and subculture medium of the present embodiment, the incubation of candidum tissue culturing seedling can be ensured when not having plant growth regulator.In addition, in the present embodiment, proliferation and subculture medium adds active carbon for adsorbing the part noxious material produced in incubation, and increases venting quality, is beneficial to root of hair.
Root media in the present embodiment, to make mother liquor by oneself as minimal medium, adds agar 6g/L in minimal medium, white sugar 20g/L, active carbon 0.5g/L, banana 50g/L, self-control natural organic liquid fertilizer 1.5g/L, bactericidal agent 2.5ml/L.In the present embodiment, the preparation process of root media is specific as follows: (1), take each component according to the self-control composition of mother liquor and consumption, makes it fully dissolve, and takes successively by respective concentration.(2), in self-control mother liquor, all kinds of material is added by required, it is respectively: agar 6g/L, white sugar 20g/L, active carbon 0.5g/L, banana 50g/L, self-control natural organic liquid fertilizer 1.5g/L, bactericidal agent 2.5ml/L, wherein: the banana in the present embodiment refers to banana puree, this banana puree is removed the peel by banana, and being put in after weighing in cooking machine adds water breaks into banana puree; The detailed process adding all kinds of material in self-control mother liquor is: distilled water heating is boiled, after getting distilled water dissolving white sugar, add agar and stir, treat that it melts completely, active carbon, banana puree, self-control natural organic liquid fertilizer, bactericidal agent, minimal medium are added simultaneously, survey pH value with pH test paper, and be 5.8 by NaOH or the HCI adjust ph of 0.1mol/L, finally adding water, it is volume required to be settled to.(3), by filling in blake bottle for the root media prepared, then seal with bottle cap, put into high-pressure steam sterilizing pan sterilizing, its sterilising temp is 120 ~ 126 DEG C, sterilization time is 10 ~ 40min, sterilized good medium is placed in condensation chamber, observe pollution-free after namely can be used for inoculation.The banana puree of adding in root media in the present embodiment has good facilitation to root system, and by other specific component as cooperation, utilize the specific prescription of rooting medium of the present invention significantly can promote root growth, self-control natural organic liquid fertilizer especially is wherein that the follow-up guarantee to survival rate serves key effect.In addition, the present embodiment, by adding bactericidal agent, reduces pollution rate.
Be described as follows about making natural organic liquid fertilizer in the present embodiment by oneself, explant induction medium is identical with the self-control natural organic liquid fertilizer formula added in root media, and the formula of this self-control natural organic liquid fertilizer is specific as follows: raw bran 3000g, lactic acid bacteria 500g, whole milk powder 500g, water 50000g, rice bran 3000g, saccharomycete 500g, phosphate fertilizer 500g, brown sugar 6000g, sea-tangle 3000g, streaky pork 500g, fishbone dust 500g, 6, egg.The manufacturing process of making natural organic liquid fertilizer in the present embodiment by oneself is: (a) breaks into powder after being dried by sea-tangle; B () is all poured all materials into fertile bucket mix and blend according to above-mentioned formula and airtightly to be sealed up for safekeeping, wherein: the rice bran in the present embodiment is fine rice bran, raw bran is made up of soya-bean cake and peanut press pulp, and soya-bean cake: peanut press pulp=1:1 (mass ratio), lactic acid bacteria is yoghurt; C () every day is sooner or later each stirs once, can be specifically morning 8:00 and evening 18:00 respectively stir once; D () treats that after fermentation in 50 ~ 60 days is become thoroughly decomposed, odorless, can use.The specific self-control natural organic liquid fertilizer of the present embodiment configuration is made up of eighties kinds of natural micro-calcics, magnesium, sulphur, copper, iron, molybdenum, manganese, aluminium, boron, zinc, lead, potassium, sodium, phosphorus, vitamin B1, vitamin C, chlorophyll, carotin etc.The research of inventor through more than ten years finds, use the vegetation period of adopting this self-control natural organic liquid fertilizer can be used in, supplement the nutritive element needed for the plant growth stage, make it obtain balanced nutrient, more fully growth promoting effects, and improve plant disease-resistant ability, the quality of plantlet in vitro is improved with this, promote the growth of root system and blade, improve transplanting survival rate, this is the key point that the present invention can realize.
Be described as follows about making mother liquor in the present embodiment by oneself, the formula of self-control mother liquor is specially: ammonium sulfate 0.46g/L, calcium chloride 0.17g/L, inositol 0.1g/L, potassium nitrate 2.83g/L, ferrous sulfate 0.03g/L, 1-methyl α-naphthyl acetate 2mg/L, potassium dihydrogen phosphate 0.4g/L, magnesium sulfate 0.19g/L.The present embodiment does not adopt MS medium as minimal medium at root media, and adopts self-control mother liquor to be key point of the present invention as minimal medium.The 1-methyl α-naphthyl acetate added in self-control mother liquor in the present embodiment may be used for the growth of inducing adventitious root, and plant is different to the demand of mineral salt at different growth and development stages, therefore the demand of different growth and development stages to mineral salt can be met in this ratio self-control mother liquor.The present embodiment adopts self-control mother liquor to improve quality and the seedling transplanting survival rate of candidum tissue culturing seedling, thus solves the key technical problems in dendrobium candidum industrialization production.
Adopt the candidum tissue culturing seedling medium of the present embodiment to cultivate, the cultural method concrete steps of candidum tissue culturing seedling are:
The bringing out and cultivate of step one, seed: choose full grains, without the dendrobium candidum fruit pod of damage by disease and insect as explant, superclean bench is tentatively pruned it, rinse 3 times with 75% ethanolic solution, then rinse 1 time with 95% ethanolic solution, be filtered dry moisture.Dendrobium candidum fruit pod is cut with aseptic cutter, seed in fruit pod is spread out on explant induction medium, induction seed germination, explant induction medium is placed on culturing rack, cultivation temperature is 23 ~ 25 DEG C, light application time is 12 hours/day, cultivates 30 ~ 45 days under intensity of illumination 1500Lx condition, and seed germination forms plumule;
Step 2, plumule proliferation and subculture are cultivated: transferred by the plumule formed in step one and to cultivate on proliferation and subculture medium, proliferation and subculture medium is placed in culturing room, cultivation temperature is 23 ~ 25 DEG C, light application time is 12 hours/day, cultivate 80 ~ 90 days under intensity of illumination 1800 ~ 2000Lx condition, obtain dendrobium candidum seedling;
The cultivation of step 3, seedling of taking root: the dendrobium candidum seedling obtained in step 2 is transferred on root media, it is cultivated, cultivation temperature is 25 ~ 26 DEG C, light application time is 12 hours/day, cultivate 100 ~ 120 days under intensity of illumination 1800 ~ 2000Lx condition, obtain dendrobium candidum strong sprout;
Step 4, transplanting: transplant after the dendrobium candidum after step 3 is carried out culture of rootage carries out hardening strong sprout.
The candidum tissue culturing seedling concrete condition that the present embodiment is cultivated is in table 1, can prove that the present embodiment passes through to add natural organic matter in the medium of candidum tissue culturing seedling thus, the quality of plantlet in vitro is improved with this, promote the growth of root system and blade, improve quality and the seedling transplanting survival rate of candidum tissue culturing seedling, thus solve the key technical problems in dendrobium candidum industrialization production.
The candidum tissue culturing seedling concrete condition of table 1 embodiment 1-3
Embodiment 1 Embodiment 2 Embodiment 3
Start rootage duration 105 ~ 110 days 115 ~ 120 days 110 ~ 120 days
Bottle outlet radical in previous existence amount Article 4 ~ 5, root system Article 3 ~ 4, root system Article 3 ~ 4, root system
Survival rate after transplanting 97% 95% 95%
Seedling strain rugosity before bottle outlet 2.5~4mm 2.5~3mm 3~3.5mm
Seedling plant height degree before bottle outlet 5~8cm 4~7cm 5~7cm
The individual plant seedling strain number of blade before bottle outlet 6 ~ 10 6 ~ 8 6 ~ 8
Embodiment 2
A kind of candidum tissue culturing seedling medium of the present embodiment, its explant induction medium, proliferation and subculture medium and root media are specific as follows: explant induction medium is using MS medium as minimal medium, agar 5g/L is added in minimal medium, white sugar 30g/L, active carbon 0.5g/L, banana 65g/L, self-control organic fertilizer 1.8g/L, bactericidal agent 2ml/L.Proliferation and subculture medium, using MS medium as minimal medium, adds agar 5g/L in minimal medium, white sugar 30g/L, active carbon 0.5g/L, banana 47g/L, bactericidal agent 2ml/L.Root media, to make mother liquor by oneself as minimal medium, adds agar 5g/L in minimal medium, white sugar 20g/L, active carbon 0.5g/L, banana 53g/L, self-control natural organic liquid fertilizer 1.6g/L.
In the present embodiment, the preparation process of explant inducing culture, proliferation and subculture medium and root media is substantially with embodiment 1, does not repeat them here.In addition, the self-control natural organic liquid fertilizer applied in the present embodiment and self-control mother liquor are with embodiment 1.Adopt the candidum tissue culturing seedling medium of the present embodiment to cultivate, the cultural method concrete steps of candidum tissue culturing seedling are with embodiment 1, and the candidum tissue culturing seedling concrete condition of cultivation is in table 1.
Embodiment 3
A kind of candidum tissue culturing seedling medium of the present embodiment, its explant induction medium, proliferation and subculture medium and root media are specific as follows: explant induction medium is using MS medium as minimal medium, agar 9g/L is added in minimal medium, white sugar 28g/L, active carbon 0.6g/L, banana 75g/L, self-control organic fertilizer 2g/L, bactericidal agent 1.5ml/L.Proliferation and subculture medium, using MS medium as minimal medium, adds agar 9g/L in minimal medium, white sugar 32g/L, active carbon 0.48g/L, banana 45g/L, bactericidal agent 1.5ml/L.Root media, to make mother liquor by oneself as minimal medium, adds agar 9g/L in minimal medium, white sugar 22g/L, active carbon 0.52g/L, banana 55g/L, self-control natural organic liquid fertilizer 1.5g/L, bactericidal agent 1.5ml/L.
In the present embodiment, the preparation process of explant inducing culture, proliferation and subculture medium and root media is substantially with embodiment 1, does not repeat them here.In addition, the self-control natural organic liquid fertilizer applied in the present embodiment and self-control mother liquor are with embodiment 1.Adopt the candidum tissue culturing seedling medium of the present embodiment to cultivate, the cultural method concrete steps of candidum tissue culturing seedling are with embodiment 1, and the candidum tissue culturing seedling concrete condition of cultivation is in table 1.
Embodiment 4
A kind of candidum tissue culturing seedling medium of the present embodiment, its explant induction medium, proliferation and subculture medium and root media are specific as follows: explant induction medium is using MS medium as minimal medium, agar 7g/L is added in minimal medium, white sugar 29g/L, active carbon 0.55g/L, banana 70g/L, self-control organic fertilizer 1.5g/L, bactericidal agent 2ml/L.Proliferation and subculture medium, using MS medium as minimal medium, adds agar 9g/L in minimal medium, white sugar 31g/L, active carbon 0.49g/L, banana 45g/L, bactericidal agent 1.5ml/L.Root media, to make mother liquor by oneself as minimal medium, adds agar 9g/L in minimal medium, white sugar 21g/L, active carbon 0.55g/L, banana 55g/L, self-control natural organic liquid fertilizer 1.8g/L, bactericidal agent 2ml/L.
In the present embodiment, the preparation process of explant inducing culture, proliferation and subculture medium and root media is substantially with embodiment 1, does not repeat them here.In addition, the self-control natural organic liquid fertilizer applied in the present embodiment and self-control mother liquor are with embodiment 1.Adopt the candidum tissue culturing seedling medium of the present embodiment to cultivate, the cultural method concrete steps of candidum tissue culturing seedling are with embodiment 1, and the candidum tissue culturing seedling concrete condition of cultivation is substantially with embodiment 1.
Although be described in detail the specific embodiment of the present invention in conjunction with specific embodiments, it is not the restriction to this patent protection domain.In claims limited range, the various amendment that those skilled in the art can make without creative work or adjustment are still by the protection of this patent.

Claims (6)

1. a cultural method for candidum tissue culturing seedling, is characterized in that, the steps include:
The bringing out and cultivate of step one, seed
Choose dendrobium candidum fruit pod as explant, dendrobium candidum fruit pod is cut with aseptic cutter, seed in fruit pod is spread out on explant induction medium, induction seed germination, be placed on culturing rack by explant induction medium, cultivation temperature is 23 ~ 25 DEG C, and light application time is 12 hours/day, cultivate 30 ~ 45 days under intensity of illumination 1500Lx condition, seed germination forms plumule;
Step 2, plumule proliferation and subculture are cultivated
The plumule formed in step one is transferred and to cultivate on proliferation and subculture medium, be placed in culturing room by proliferation and subculture medium, cultivation temperature is 23 ~ 25 DEG C, and light application time is 12 hours/day, cultivate 80 ~ 90 days under intensity of illumination 1800 ~ 2000Lx condition, obtain dendrobium candidum seedling;
The cultivation of step 3, seedling of taking root
Be transferred on root media by the dendrobium candidum seedling obtained in step 2, cultivate it, cultivation temperature is 25 ~ 26 DEG C, and light application time is 12 hours/day, cultivates 100 ~ 120 days under intensity of illumination 1800 ~ 2000Lx condition, obtains dendrobium candidum strong sprout;
Step 4, transplanting
Transplant after dendrobium candidum after step 3 is carried out culture of rootage carries out hardening strong sprout.
2. the cultural method of a kind of candidum tissue culturing seedling according to claim 1, it is characterized in that, explant induction medium in step one is using MS medium as minimal medium, agar 5 ~ 9g/L is added in minimal medium, white sugar 28 ~ 30g/L, active carbon 0.5 ~ 0.6g/L, banana 65 ~ 75g/L, self-control organic fertilizer 1.5 ~ 2g/L, bactericidal agent 1.5 ~ 2.5ml/L, the pH of explant induction medium is 5.4 ~ 5.8, disinfecting action is carried out to above-mentioned explant induction medium, its sterilising temp is 120 ~ 126 DEG C, sterilization time is 10 ~ 40min.
3. the cultural method of a kind of candidum tissue culturing seedling according to claim 1, it is characterized in that, proliferation and subculture medium in step 2 is using MS medium as minimal medium, agar 5 ~ 9g/L is added in minimal medium, white sugar 30 ~ 32g/L, active carbon 0.48 ~ 0.5g/L, banana 45 ~ 50g/L, bactericidal agent 1.5 ~ 2.5ml/L, the pH of proliferation and subculture medium is 5.4 ~ 5.8, carry out disinfecting action to above-mentioned proliferation and subculture medium, its sterilising temp is 120 ~ 126 DEG C, and sterilization time is 10 ~ 40min.
4. the cultural method of a kind of candidum tissue culturing seedling according to claim 2, it is characterized in that, root media in step 3 is to make mother liquor by oneself as minimal medium, agar 5 ~ 9g/L is added in minimal medium, white sugar 20 ~ 22g/L, active carbon 0.5 ~ 0.55g/L, banana 50 ~ 55g/L, self-control natural organic liquid fertilizer 1.5 ~ 1.8g/L, bactericidal agent 1.5 ~ 2.5ml/L, the pH of root media is 5.4 ~ 5.8, carries out disinfecting action to above-mentioned root media, its sterilising temp is 120 ~ 126 DEG C, and sterilization time is 10 ~ 40min.
5. the cultural method of a kind of candidum tissue culturing seedling according to claim 4, it is characterized in that, described explant induction medium is identical with the self-control natural organic liquid fertilizer formula added in root media, and the formula of this self-control natural organic liquid fertilizer is specific as follows: raw bran 3000g, lactic acid bacteria 500g, whole milk powder 500g, water 50000g, rice bran 3000g, saccharomycete 500g, phosphate fertilizer 500g, brown sugar 6000g, sea-tangle 3000g, streaky pork 500g, fishbone dust 500g, 6, egg.
6. the cultural method of a kind of candidum tissue culturing seedling according to claim 4 or 5, it is characterized in that, the formula of the self-control mother liquor in described root media is specially: ammonium sulfate 0.46g/L, calcium chloride 0.17g/L, inositol 0.1g/L, potassium nitrate 2.83g/L, ferrous sulfate 0.03g/L, 1-methyl α-naphthyl acetate 2mg/L, potassium dihydrogen phosphate 0.4g/L, magnesium sulfate 0.19g/L.
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CN106561247A (en) * 2016-11-10 2017-04-19 唐翔 Cultivation method for imitated wild Dendrobium nobile Lindl
CN106962205A (en) * 2017-06-02 2017-07-21 合肥华盖生物科技有限公司 A kind of photinia glabra tissue-cultured seedling root media and cultural method

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