CN103314858A - Seed tissue culture propagation method of dendrobium candidum - Google Patents
Seed tissue culture propagation method of dendrobium candidum Download PDFInfo
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Abstract
The invention relates to a seed tissue culture propagation method of dendrobium candidum and belongs to the technical field of planting of traditional Chinese medicinal materials. The seed tissue culture propagation method of dendrobium candidum comprises the following four steps of: introduction and sterilization, induction culture, differentiation culture as well as strong seedling and rooting culture. The seed tissue culture propagation method of dendrobium candidum has the beneficial effects that seedlings obtained by seed tissue culture are difficult to degenerate, genetic traits are stable, active substance content is close to that of a wild variety, and quality of seeds is guaranteed; quantity of the seeds of dendrobium candidum is high, and propagation quantity of the seeds can be greatly increased; the seed tissue culture propagation method of dendrobium candidum also greatly shortens a tissue culture propagation period of dendrobium candidum, the tissue culture period from seed shearing to rooting and seedling growing is only 4-5 months, production time is greatly shortened, and production cost is reduced.
Description
Technical field
The invention belongs to the seed tissue culture propagation method in traditional Chinese medicine planting technology field, particularly a kind of dendrobium candidum.
Background technology
The stem of noble dendrobium is China's tradition rare traditional Chinese medicine, and it is all on the books that edition pharmacopeia is gone through by China.The former plant of the Chinese medicine stem of noble dendrobium be the orchid family (Orchidaceae) Dendrobium (
DendrobiumSw.) herbaceos perennial.Dendrobium is one of larger genus of the orchid family, and the whole world approximately has more than 1000 to plant, and China's Dendrobium Sw has 74 kind of 2 mutation, and wherein accounting for of circulation of commodities more than 50 planted.Wherein precious with dendrobium candidum, be the superfine product of all stems of noble dendrobium.Dendrobium candidum is endangered species, belongs to national secondary and lays special stress on protecting plant, and be famous and precious rare traditional Chinese medicine.Ancient times, the noble claimed that dendrobium candidum is " purple principal columns of a hall celestial being is pretty ", " Qian Nianrun ", claim again " the celestial grass of help " among the people.Authentic dendrobium candidum is the iron green, and gas is little, and that chews has a stickiness, distinguish the flavor of sweet, and few slag eats without any side effects for a long time.Be twisted into helical form and the rear title of oven dry while frying with dendrobium candidum
Tiepi Fengdou.
For many years, the commodity stem of noble dendrobium mainly relies on the Wild Medicinal Dendrobium Plants, and along with deepening continuously and the year by year increase of demand both at home and abroad of medicinal dendrobium development and use, China's Wild Medicinal Dendrobium Plants has suffered heavy damage, some area even face exhaustion.In order to protect Wild Orchids Resources; each state has all put into effect many statutes and regulations of forbidding excavating or selling Wild Orchids; in " the Conservation of resource management rules " of State Council's appearance in 1987, dendrobium loddigesii Rolfe, HERBA DENDROBII, HERBA DENDROBII, stem of Eyeshaped Dendrobium and dendrobium candidum are listed in three grades of Precious, Rare, Endangered protective plants of country.People begin gradually wild plant to dendrobium candidum and carry out house and plant cultivation, high-yield culture technique etc. and put into practice thus.The propagation technique of dendrobium candidum is mainly seminal propagation, division propagation and cottage propagation dual mode at present.
Collect because Seeds of Dendrobium Candidum is difficult, percentage of seedgermination is low, and seminal propagation, and seedling raise period is long, has greatly increased the cultivation cost of dendrobium candidum, is unfavorable for the artificial establishing in large scale of dendrobium candidum.So in current artificial cultivation, breeding is main to rely on division propagation and cottage propagation, but the method reproduction coefficient is low, both uneconomical, limited again the yield potentiality of dendrobium candidum, inconvenient Planting management is promoted, thereby the expansion cultivated area is suppressed.People have begun artificial propagation, and many places have adopted tissue culture propagation method with a large amount of seedling of quick acquisition; Reproduction speed is very slow at present, can't satisfy actual needs.
Summary of the invention
The object of the present invention is to provide a kind of seed tissue culture propagation method of dendrobium candidum, to improve the reproduction speed of dendrobium candidum, realize standardization and the large-scale development of officinal dendrobium stem plantation; For realizing this purpose, the inventor is through long term test, various tissue culture prescriptions and the cultural method of breeding dendrobium candidum have successfully been studied, to tissue culture prescription and production technology, and the special habit of growth of dendrobium candidum carried out large quantity research and repetition test, obtained culture medium prescription and the cultural method of original creation.
For solving the problems of the technologies described above, the technical solution adopted in the present invention is the seed tissue culture propagation method of dendrobium candidum, comprise introduce a fine variety sterilization, induce cultivation, differentiation cultivation, strong sprout and culture of rootage four large steps, it is characterized in that concrete grammar is as follows:
⑴ introduce a fine variety sterilization: choose uncracked wild dendrobium candidum ripening fruits, wash under flowing water, put into the 1% liquid detergent aqueous solution and stir 5～8min, use aseptic water washing 10～12min again; On superclean bench, fruit is placed in 70% the alcohol sterilization 20 seconds, and used again 0.1% HgCl
2Then aqueous solution soaking 7～9min uses aseptic water washing 5～6 times.
⑵ induce cultivation: the fruit after will disinfecting cuts epidermis in gnotobasis, fruit is cut into the square of 0.1mm, put into sterile water and rock, the medication spoon is seeded on the inducing culture 3～5 of every bottle graft kinds after the vibration evenly, in 22 ℃ of temperature at night, warm 24 ℃ of day, relative moisture 68%～70% is carried out Seed inducement and is cultivated under the condition of unglazed photograph, after 15～20 days, seed begins to sprout and expands; Continue to cultivate after 10 days, can see the protocorm blank of loose shape; Be transferred to protocorm in the short shape medium this moment, in 23 ℃ of temperature at night, warm 25 ℃ of day, relative moisture 70%～72%, intensity of illumination 1600lx, the morning illumination time started every day 7:00, the short shape of protocorm of carrying out seed germination under 14 hours the condition of light irradiation time every day is cultivated, and after 25～30 days, the protocorm blank surface of loose shape forms rough particle gradually, along with the prolongation of incubation time, particle is grown up and is formed the protocorm particle of diameter 1～2mm.
⑶ break up cultivation: the protocorm that growth conditions is good is transferred on the differential medium, 45～50 of every bottle graft kinds, in 25 ℃ of temperature at night, warm 27 ℃ of day, relative moisture 73%～77%, intensity of illumination 1800lx, morning illumination time started every day, 7:00 broke up cultivation under 12 hours the condition of light irradiation time every day, within 20～25 days, can be observed protocorm begin the differentiation sprout a little, then temperature is adjusted into 26 ℃ of temperature at night, warm 28 ℃ of day, within the 35th～40 day, can be observed protocorm and grow budlet and begin leaflet, can see grow tall 1～1.5cm and with 2～3 leaflets of propagation bud by the 50th day, obtain breaking up seedling.
⑷ strong sprout and culture of rootage: will break up the plant height 1～2cm after cultivating, test-tube plantlet with 2～3 leaves is inoculated on the root media, every bottle graft kind 10～15 strains, in 26 ℃ of temperature at night, warm 28 ℃ of day, relative moisture 73%～77%, intensity of illumination 2000lx, morning illumination time started every day, 7:00 carried out culture of rootage under 12 hours the condition of light irradiation time every day, within the 20th～25 day, base portion begins to occur the long white root of 2～3 treaty 1cm, every young plant has 3～5 roots, and subsequently gradually growth is within the 35th～40 day, rooting rate reaches more than 90%, coring length 1.5cm～2.0cm, height of seedling 3～5cm namely obtains the seedling of taking root of dendrobium candidum, at this moment can carry out hardening, transplant.
In the above-mentioned tissue culture propagation method, the prescription of each stage medium of mentioning is:
Inducing culture: N6+NAA0.18mg/L+20% coconut palm breast+2% sucrose+active carbon 200mg/L; PH=5.8.
Short shape medium: N6+NAA0.25mg/L+20% coconut palm breast+2% sucrose+active carbon 200mg/L; PH=5.8.
Differential medium: N6+6～BA0.53mg/L+NAA0.20mg/L+10% bananas juice+2% sucrose+active carbon 200mg/L; PH=5.8.
Root media: N6+IBA0.5mg/L+10% bananas juice+2% sucrose+active carbon 200mg/L; PH=5.6.
The invention has the beneficial effects as follows and adopt seed group to train resulting seedling, be difficult for degenerating, hereditary dimensionally stable, active principle content is close with wild kind, has guaranteed the quality of seed; The seed amount of dendrobium candidum is large, can greatly increase the breeding quantity of seed; The inventive method has also shortened the tissue culture propagation cycle of dendrobium candidum greatly simultaneously, and the group training cycle from the shearing seed to the seedling of taking root only needs 4～5 months, has greatly shortened the production time, has reduced production cost.
Embodiment
The invention will be further described below in conjunction with embodiment, and following examples are intended to illustrate the present invention rather than limitation of the invention further, should not limit protection scope of the present invention with this.
Embodiment 1.
At first prepare the medium material:
Inducing culture: N6+NAA0.18mg/L+20% coconut palm breast+2% sucrose+active carbon 200mg/L; PH=5.8.
Short shape medium: N6+NAA0.25mg/L+20% coconut palm breast+2% sucrose+active carbon 200mg/L; PH=5.8.
Differential medium: N6+6～BA0.53mg/L+NAA0.20mg/L+10% bananas juice+2% sucrose+active carbon 200mg/L; PH=5.8.
Root media: N6+IBA0.5mg/L+10% bananas juice+2% sucrose+active carbon 200mg/L; PH=5.6.
Choose uncracked wild dendrobium candidum ripening fruits, under flowing water, wash, put into the 1% liquid detergent aqueous solution and stir 5min, use again aseptic water washing 10min; On superclean bench, fruit is placed in 70% the alcohol sterilization 20 seconds, and used again 0.1% HgCl
2Then aqueous solution soaking 7min uses aseptic water washing 5～6 times; Then fruit is cut epidermis in gnotobasis, fruit is cut into the square of 0.1mm, put into sterile water and rock, the medication spoon is seeded on the inducing culture 3 of every bottle graft kinds after the vibration evenly, in 22 ℃ of temperature at night, warm 24 ℃ of day, relative moisture 68% is carried out Seed inducement and is cultivated under the condition of unglazed photograph, after 15～20 days, seed begins to sprout and expands; Continue to cultivate after 10 days, can see the protocorm blank of loose shape; Be transferred to protocorm in the short shape medium this moment, in 23 ℃ of temperature at night, warm 25 ℃ of day, relative moisture 70%, intensity of illumination 1600lx, the morning illumination time started every day 7:00, the short shape of protocorm of carrying out seed germination under 14 hours the condition of light irradiation time every day is cultivated, and after 25～30 days, the protocorm blank surface of loose shape forms rough particle gradually, along with the prolongation of incubation time, particle is grown up and is formed the protocorm particle of diameter 1～2mm; Then the protocorm that growth conditions is good is transferred on the differential medium, 45 of every bottle graft kinds, in 25 ℃ of temperature at night, warm 27 ℃ of day, relative moisture 73%, intensity of illumination 1800lx, morning illumination time started every day, 7:00 broke up cultivation under 12 hours the condition of light irradiation time every day, within 20～25 days, can be observed protocorm begin the differentiation sprout a little, then temperature is adjusted into 26 ℃ of temperature at night, warm 28 ℃ of day, within the 35th～40 day, can be observed protocorm and grow budlet and begin leaflet, can see grow tall 1～1.5cm and with 2～3 leaflets of propagation bud by the 50th day, obtain breaking up seedling; Then will break up the plant height 1～2cm after cultivating, test-tube plantlet with 2～3 leaves is inoculated on the root media, every bottle graft kind 10 strains, in 26 ℃ of temperature at night, warm 28 ℃ of day, relative moisture 73%, intensity of illumination 2000lx, the morning illumination time started every day 7:00, carry out culture of rootage under 12 hours the condition of light irradiation time every day, within the 20th～25 day, base portion begins to occur the long white root of 2～3 treaty 1cm, and every young plant has 3～5 roots, subsequently gradually growth, to within the 35th～40 day, rooting rate reaches more than 90%, coring length 1.5cm～2.0cm, height of seedling 3～5cm namely obtains the seedling of taking root of dendrobium candidum.
Embodiment 2.
Choose uncracked wild dendrobium candidum ripening fruits, under flowing water, wash, put into the 1% liquid detergent aqueous solution and stir 7min, use again aseptic water washing 11min; On superclean bench, fruit is placed in 70% the alcohol sterilization 20 seconds, and used again 0.1% HgCl
2Then aqueous solution soaking 8min uses aseptic water washing 5～6 times; Then fruit is cut epidermis in gnotobasis, fruit is cut into the square of 0.1mm, put into sterile water and rock, the medication spoon is seeded on the inducing culture 4 of every bottle graft kinds after the vibration evenly, in 22 ℃ of temperature at night, warm 24 ℃ of day, relative moisture 69% is carried out Seed inducement and is cultivated under the condition of unglazed photograph, after 15～20 days, seed begins to sprout and expands; Continue to cultivate after 10 days, can see the protocorm blank of loose shape; Be transferred to protocorm in the short shape medium this moment, in 23 ℃ of temperature at night, warm 25 ℃ of day, relative moisture 71%, intensity of illumination 1600lx, the morning illumination time started every day 7:00, the short shape of protocorm of carrying out seed germination under 14 hours the condition of light irradiation time every day is cultivated, and after 25～30 days, the protocorm blank surface of loose shape forms rough particle gradually, along with the prolongation of incubation time, particle is grown up and is formed the protocorm particle of diameter 1～2mm; Then the protocorm that growth conditions is good is transferred on the differential medium, 50 of every bottle graft kinds, in 25 ℃ of temperature at night, warm 27 ℃ of day, relative moisture 75%, intensity of illumination 1800lx, morning illumination time started every day, 7:00 broke up cultivation under 12 hours the condition of light irradiation time every day, within 20～25 days, can be observed protocorm begin the differentiation sprout a little, then temperature is adjusted into 26 ℃ of temperature at night, warm 28 ℃ of day, within the 35th～40 day, can be observed protocorm and grow budlet and begin leaflet, can see grow tall 1～1.5cm and with 2～3 leaflets of propagation bud by the 50th day, obtain breaking up seedling; Then will break up the plant height 1～2cm after cultivating, test-tube plantlet with 2～3 leaves is inoculated on the root media, every bottle graft kind 15 strains, in 26 ℃ of temperature at night, warm 28 ℃ of day, relative moisture 75%, intensity of illumination 2000lx, the morning illumination time started every day 7:00, carry out culture of rootage under 12 hours the condition of light irradiation time every day, within the 20th～25 day, base portion begins to occur the long white root of 2～3 treaty 1cm, and every young plant has 3～5 roots, subsequently gradually growth, to within the 35th～40 day, rooting rate reaches more than 90%, coring length 1.5cm～2.0cm, height of seedling 3～5cm namely obtains the seedling of taking root of dendrobium candidum.
Embodiment 3.
Choose uncracked wild dendrobium candidum ripening fruits, under flowing water, wash, put into the 1% liquid detergent aqueous solution and stir 8min, use again aseptic water washing 12min; On superclean bench, fruit is placed in 70% the alcohol sterilization 20 seconds, and used again 0.1% HgCl
2Then aqueous solution soaking 9min uses aseptic water washing 5～6 times; Then fruit is cut epidermis in gnotobasis, fruit is cut into the square of 0.1mm, put into sterile water and rock, the medication spoon is seeded on the inducing culture 5 of every bottle graft kinds after the vibration evenly, in 22 ℃ of temperature at night, warm 24 ℃ of day, relative moisture 70% is carried out Seed inducement and is cultivated under the condition of unglazed photograph, after 15～20 days, seed begins to sprout and expands; Continue to cultivate after 10 days, can see the protocorm blank of loose shape; Be transferred to protocorm in the short shape medium this moment, in 23 ℃ of temperature at night, warm 25 ℃ of day, relative moisture 72%, intensity of illumination 1600lx, the morning illumination time started every day 7:00, the short shape of protocorm of carrying out seed germination under 14 hours the condition of light irradiation time every day is cultivated, and after 25～30 days, the protocorm blank surface of loose shape forms rough particle gradually, along with the prolongation of incubation time, particle is grown up and is formed the protocorm particle of diameter 1～2mm; Then the protocorm that growth conditions is good is transferred on the differential medium, 50 of every bottle graft kinds, in 25 ℃ of temperature at night, warm 27 ℃ of day, relative moisture 77%, intensity of illumination 1800lx, morning illumination time started every day, 7:00 broke up cultivation under 12 hours the condition of light irradiation time every day, within 20～25 days, can be observed protocorm begin the differentiation sprout a little, then temperature is adjusted into 26 ℃ of temperature at night, warm 28 ℃ of day, within the 35th～40 day, can be observed protocorm and grow budlet and begin leaflet, can see grow tall 1～1.5cm and with 2～3 leaflets of propagation bud by the 50th day, obtain breaking up seedling; Then will break up the plant height 1～2cm after cultivating, test-tube plantlet with 2～3 leaves is inoculated on the root media, every bottle graft kind 15 strains, in 26 ℃ of temperature at night, warm 28 ℃ of day, relative moisture 77%, intensity of illumination 2000lx, the morning illumination time started every day 7:00, carry out culture of rootage under 12 hours the condition of light irradiation time every day, within the 20th～25 day, base portion begins to occur the long white root of 2～3 treaty 1cm, and every young plant has 3～5 roots, subsequently gradually growth, to within the 35th～40 day, rooting rate reaches more than 90%, coring length 1.5cm～2.0cm, height of seedling 3～5cm namely obtains the seedling of taking root of dendrobium candidum.
Claims (1)
1. the seed tissue culture propagation method of dendrobium candidum, comprise introduce a fine variety sterilization, induce cultivation, differentiation cultivation, strong sprout and culture of rootage four large steps, it is characterized in that concrete grammar is as follows:
⑴ introduce a fine variety sterilization: choose uncracked wild dendrobium candidum ripening fruits, wash under flowing water, put into the 1% liquid detergent aqueous solution and stir 5～8min, use aseptic water washing 10～12min again; On superclean bench, fruit is placed in 70% the alcohol sterilization 20 seconds, and used again 0.1% HgCl
2Then aqueous solution soaking 7～9min uses aseptic water washing 5～6 times;
⑵ induce cultivation: the fruit after will disinfecting cuts epidermis in gnotobasis, fruit is cut into the square of 0.1mm, put into sterile water and rock, the medication spoon is seeded on the inducing culture 3～5 of every bottle graft kinds after the vibration evenly, in 22 ℃ of temperature at night, warm 24 ℃ of day, relative moisture 68%～70% is carried out Seed inducement and is cultivated under the condition of unglazed photograph, after 15～20 days, seed begins to sprout and expands; Continue to cultivate after 10 days, can see the protocorm blank of loose shape; Be transferred to protocorm in the short shape medium this moment, in 23 ℃ of temperature at night, warm 25 ℃ of day, relative moisture 70%～72%, intensity of illumination 1600lx, the morning illumination time started every day 7:00, the short shape of protocorm of carrying out seed germination under 14 hours the condition of light irradiation time every day is cultivated, and after 25～30 days, the protocorm blank surface of loose shape forms rough particle gradually, along with the prolongation of incubation time, particle is grown up and is formed the protocorm particle of diameter 1～2mm;
⑶ break up cultivation: the protocorm that growth conditions is good is transferred on the differential medium, 45～50 of every bottle graft kinds, in 25 ℃ of temperature at night, warm 27 ℃ of day, relative moisture 73%～77%, intensity of illumination 1800lx, morning illumination time started every day, 7:00 broke up cultivation under 12 hours the condition of light irradiation time every day, within 20～25 days, can be observed protocorm begin the differentiation sprout a little, then temperature is adjusted into 26 ℃ of temperature at night, warm 28 ℃ of day, within the 35th～40 day, can be observed protocorm and grow budlet and begin leaflet, can see grow tall 1～1.5cm and with 2～3 leaflets of propagation bud by the 50th day, obtain breaking up seedling;
⑷ strong sprout and culture of rootage: will break up the plant height 1～2cm after cultivating, test-tube plantlet with 2～3 leaves is inoculated on the root media, every bottle graft kind 10～15 strains, in 26 ℃ of temperature at night, warm 28 ℃ of day, relative moisture 73%～77%, intensity of illumination 2000lx, morning illumination time started every day, 7:00 carried out culture of rootage under 12 hours the condition of light irradiation time every day, within the 20th～25 day, base portion begins to occur the long white root of 2～3 treaty 1cm, every young plant has 3～5 roots, and subsequently gradually growth is within the 35th～40 day, rooting rate reaches more than 90%, coring length 1.5cm～2.0cm, height of seedling 3～5cm namely obtains the seedling of taking root of dendrobium candidum, at this moment can carry out hardening, transplant;
In the above-mentioned tissue culture propagation method, the prescription of each stage medium of mentioning is:
Inducing culture: N6+NAA0.18mg/L+20% coconut palm breast+2% sucrose+active carbon 200mg/L; PH=5.8;
Short shape medium: N6+NAA0.25mg/L+20% coconut palm breast+2% sucrose+active carbon 200mg/L; PH=5.8;
Differential medium: N6+6～BA0.53mg/L+NAA0.20mg/L+10% bananas juice+2% sucrose+active carbon 200mg/L; PH=5.8;
Root media: N6+IBA0.5mg/L+10% bananas juice+2% sucrose+active carbon 200mg/L; PH=5.6.
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| CN107251728A (en) * | 2017-05-09 | 2017-10-17 | 贵州济生农业科技有限公司 | A kind of extracting method of dendrobium candidum |
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