CN104429972A - Explant induction culture medium for dendrobium officinale tissue culture seedling culture - Google Patents
Explant induction culture medium for dendrobium officinale tissue culture seedling culture Download PDFInfo
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Abstract
The invention discloses an explant induction culture medium for dendrobium officinale tissue culture seedling culture, and belongs to the technical field of plant tissue culture. The explant induction culture medium is used for spreading seeds in dendrobium officinale capsules, induces the seeds to sprout and form embryos and uses an MS culture medium as a basic culture medium, 5-9g/L of agar, 28-30g/L of white sugar, 0.5-0.6g/L of activated carbon, 65-75g/L of bananas, 1.5-2g/L of self-made organic liquid fertilizers and 1.5-2.5ml/L of sterilizing agents are added into the basic culture medium, and the pH (potential of hydrogen) of the explant induction culture medium ranges from 5.4 to 5.8. Plant growth regulators are not added into the explant induction culture medium, proliferation and differentiation of cells and tissues can be promoted by the aid of natural banana puree and the specific self-made organic liquid fertilizers, and safety and environmental protection are realized. Besides, increase of dendrobium officinale tissue culture seedling reproduction coefficient and transplanting survival rate is ensured, and the problems of slow rooting and low survival rate in the prior art are solved.
Description
Technical field
The invention belongs to field of plant tissue culture technique, more particularly, relate to a kind of explant induction medium cultivated for 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 explant induction 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 explant induction medium cultivated for candidum tissue culturing seedling, adopt technical scheme of the present invention, the reproduction restraint of biological cells and tissues can be promoted.
2. technical scheme
For achieving the above object, technical scheme provided by the invention is:
A kind of explant induction medium cultivated for candidum tissue culturing seedling of the present invention, this explant induction medium is for disseminating the seed in dendrobium candidum fruit pod, and induction seed germination forms plumule.
Further, described explant induction medium 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.
Further, the formula making natural organic liquid fertilizer by oneself 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, carry out disinfecting action to explant inducing culture, its sterilising temp is 120 ~ 126 DEG C, and sterilization time is 10 ~ 40min.
3. beneficial effect
Adopt technical scheme provided by the invention, compared with prior art, there is following remarkable result:
A kind of explant induction medium cultivated for candidum tissue culturing seedling of the present invention, plant growth regulator is not added in this explant induction medium, adopt natural banana puree and specific self-control organic fertilizer can promote the reproduction restraint of biological cells and tissues, safe and environmentally friendly.The trace element that the present invention makes by oneself in organic fertilizer is various, can be more balanced be beneficial to plant growth, for the reproduction coefficient and transplanting survival rate that improve candidum tissue culturing seedling provide guarantee, the problem that what the cultural method solving traditional dendrobium candidum brought take root slowly, survival rate is not high, the medium that the candidum tissue culturing seedling cultivated does not add natural organic matter cultivate seedling strain rugosity, highly, the number of blade all has increases and have good facilitation to root system.
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 explant induction medium cultivated for candidum tissue culturing seedling of the present embodiment, this explant induction medium is for disseminating the seed in dendrobium candidum fruit pod, and induction seed germination forms plumule.Concrete explant induction medium, using MS medium as minimal medium, adds agar 5.5g/L in minimal medium, white sugar 30g/L, active carbon 0.5g/L, banana 67g/L in the present embodiment, 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.
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.
Adopt a kind of explant induction medium for candidum tissue culturing seedling cultivation of the present embodiment to cultivate, carrying out cultivation candidum tissue culturing seedling concrete steps is:
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 disseminated on explant induction medium in the present embodiment, 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.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.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.
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.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.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 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 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.
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 explant induction medium of the present embodiment can improve the quality of plantlet in vitro thus, 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 explant induction medium cultivated for candidum tissue culturing seedling of the present embodiment, this 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.Adopt the explant induction 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 explant induction medium cultivated for candidum tissue culturing seedling of the present embodiment, this 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.Adopt the explant induction 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 explant induction medium cultivated for candidum tissue culturing seedling of the present embodiment, this 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.Adopt the explant induction 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 (4)
1. for the explant induction medium that candidum tissue culturing seedling is cultivated, it is characterized in that, this explant induction medium is for disseminating the seed in dendrobium candidum fruit pod, and induction seed germination forms plumule.
2. a kind of explant induction medium cultivated for candidum tissue culturing seedling according to claim 1, it is characterized in that, described explant induction medium is using MS medium as minimal medium, agar 5 ~ 9g/L is added, white sugar 28 ~ 30g/L, active carbon 0.5 ~ 0.6g/L in minimal medium, 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.
3. a kind of explant induction medium cultivated for candidum tissue culturing seedling according to claim 2, it is characterized in that, the formula of 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.
4. a kind of explant induction medium cultivated for candidum tissue culturing seedling according to claim 2, it is characterized in that, carry out disinfecting action to explant inducing culture, its sterilising temp is 120 ~ 126 DEG C, and sterilization time is 10 ~ 40min.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106365741A (en) * | 2016-08-30 | 2017-02-01 | 柳州市泓吉农业科技有限公司 | Foliage fertilizer for dendrobium officinale |
| CN106359091A (en) * | 2016-08-30 | 2017-02-01 | 柳州市泓吉农业科技有限公司 | Culture medium for dendrobium officinale tissue culture seedling |
| CN106396767A (en) * | 2016-08-30 | 2017-02-15 | 柳州市泓吉农业科技有限公司 | Leaf surface spraying fertilizer for dendrobium officinale |
| CN106396768A (en) * | 2016-08-30 | 2017-02-15 | 柳州市泓吉农业科技有限公司 | Yield increasing fertilizer for dendrobium officinale |
| CN114831027A (en) * | 2022-05-25 | 2022-08-02 | 广西壮族自治区中国科学院广西植物研究所 | Dendrobium devonianum tissue culture rapid propagation culture medium combination and application thereof in dendrobium devonianum seedling cultivation |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114831027A (en) * | 2022-05-25 | 2022-08-02 | 广西壮族自治区中国科学院广西植物研究所 | Dendrobium devonianum tissue culture rapid propagation culture medium combination and application thereof in dendrobium devonianum seedling cultivation |
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