CN101461328A - High-efficient propagation method of seedlings of Dendrobium officinale - Google Patents
High-efficient propagation method of seedlings of Dendrobium officinale Download PDFInfo
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Abstract
The invention relates to a propagating method of Dendrobium officinale Kimura et Migo seedlings, characterized in that, vigorous Dendrobium officinale Kimura et Migo maternal plant is performed with artificial pollination, the obtained fruit is sanitized and cut, seeds are used for bourgeoning culture medium, causing the seeds to bourgeon protocorm and further form young seedling, then the young seedling propagated in s test tube is cultivated by test tube blossom culture medium, the vigorous plant is selected with artificial pollination in the test tube after blossom, cut after fruit ripening with aseptic seeding, causing the seeds to develop into small plant, which is cultivated on good seedling culture medium, mature shoot is formed, the mature shoot is performed with seedling exercising in the natural light, furthermore cultivated into the seedling. The invention is simple in operation, low in cost, greatly shortened in maturing stage of the fruit of Dendrobium officinale Kimura et Migo, high in efficiency and no plant diseases and insect pests, and transplanting success ratio reaches more than 95%.
Description
Technical field
The present invention relates to a kind of propagation method of medicinal plant seedling, relate in particular to a kind of dendrobium candidum (Dendrobiumofficinale Kimura et Migo) reaches efficient breeding seedling by the test tube flowering and the result that pollinates method.
Background technology
Effects such as dendrobium candidum (Dendrobium officinale Kimura et Migo) is China's rare Chinese medicine commonly used, and it has nourishing Yin and clearing heat, the beneficial stomach that promotes the production of body fluid, moisten the lung and relieve the cough are usually used in fidgety, the various disease conditions such as abnormal heat after being ill of consumption of body fluid caused by febrile disease, dry.Modern pharmacological research shows, the effect that the stem of noble dendrobium also has is antitumor, anti-ageing, strengthen body immunity and hemangiectasis.But because long-term immoderate the excavating of people makes depletion of natural resources, the reproduction rate of adding dendrobium candidum itself is extremely low, and existing dendrobium candidum becomes one of medicinal material kind of special-protection-by-the-State.Therefore the artificial planting dendrobium candidum has broad prospects, and is one of bottleneck of dendrobium candidum plant husbandry but lack high quality seedling.
Summary of the invention
The objective of the invention is to develop a kind of method that can breed seedlings of Dendrobium officinale expeditiously.
The fruit seed germination medium culture that we obtain by the dendrobium candidum maternal plant artificial pollination with robust growth, seed germination becomes protocorm and further forms seedling, with seedling test tube flowering medium culture, plant artificial pollination after will blooming, aseptic seeding behind the fruit maturation, make seed grow up to plantlet, plantlet is cultivated on the strong seedling culture base, the Cheng Miao that forms just can further cultivate into seedling through hardening, the survival rate of Cheng Miao can reach more than 95%, thereby has realized purpose of the present invention.
Seedlings of Dendrobium officinale propagation method of the present invention, its feature comprises the steps:
Dendrobium candidum (the Dendrobium officinale Kimura et Migo) maternal plant of choosing robust growth when (1) blooming carries out artificial pollination, pollination back fruit maturation, with fruit as explant;
(2) explant is used successively alcohol-pickled, mercuric chloride solution sterilization is cut after the rinsed with sterile water, Powdered embryo is inoculated into the seed germination medium, seed germination forms protocorm, and further forms seedling, and described seed germination medium contains for every liter spends precious No. 1 1～1.5g, peptone 0.5～2g, coconut milk 50～100mL, sucrose 15～30g, agar 6～7g, active carbon 0.5～1g, all the other are vitamin and inositol composition in the MS medium;
(3) seedling is used the test tube flowering medium culture, the plant of choosing robust growth after blooming carries out artificial pollination in vitro, cut behind the fruit maturation and carry out aseptic seeding, seed germination forms protocorm, and further form complete plantlet, described test tube flowering medium is every liter and contains 6-benzyl purine 0.5～1.0mg, methyl 0.1～0.2mg, and all the other are the MS medium;
(4) plantlet is transplanted on the strong seedling culture base is cultivated, be formed into seedling, described strong seedling culture base contains for every liter spends precious No. 1 1～1.5g, spend precious No. 2 1～1.5g, peptone 0.5～2g, caseinhydrolysate 0.1～0.3g, bananas juice 50～150g, carrot juice 10～30g, apple juice 10～30g, sucrose 10～20g, agar 6～7g, active carbon 1～2g, methyl 0.5～1.0mg, all the other are vitamin and inositol composition in the MS medium;
(5) will become seedling at the natural daylight lower refining seedling, clean the medium of root then, in sphagna or peat and the Lan Shi matrix that 1:1 mixes by volume, cultivate, obtain seedling;
Medium pH 5.2～5.4 in above-mentioned steps (2)～(4), cultivation temperature (26 ± 2) ℃.
The described fructescence of step (1) is 110～130 days.
The described alcohol concentration of step (2) is volume fraction 70%～80% preferably, soak time preferably 30～60 seconds, the concentration of described mercuric chloride solution is mass fraction 0.1%～0.2% preferably, disinfecting time preferably 20～30 minutes, the rinsing number of times of described sterile water preferably 4～5 times, the described treasured of spending can be buied for No. 1 from the market, in order to obtain more seedling in process of production, described protocorm can also be formed the line and staff control of bud and protocorms, on the shoot proliferation medium, can carry out shoot proliferation, the protocorms that forms can be proceeded propagation, the seedling of band root then enters the described cultivation of step (3), and described shoot proliferation medium contains for every liter spends precious No. 1 1～2g, peptone 0.5～2g, coconut milk 50～100mL, sucrose 10～20g, agar 6～7g, 6-benzyl purine 0.5～1.0mg, active carbon 1～2g, all the other are vitamin and inositol composition in the MS medium.
The height of the described seedling of step (3) is preferably 2～3cm, and the described fructescence is 50～60 days.
The described one-tenth seedling of step (4) is high 3～4cm preferably, and every clump has 2～3 buds, described spending precious No. 2
The described hardening time of step (5) is preferably 8～12 days, and the illumination of described natural lighting is 1500～20001x, best 12～16 hours/day of light application time.
That uses in step (2) and (4) medium spends No. 1, treasured and spends treasured all to be commercially available for No. 2.
Though patent and the bibliographical information that produce relevant for seedlings of Dendrobium officinale domestic morning are not but seen and are utilized pollinate result's method of test tube flowering to breed dendrobium candidum.And method of the present invention, not only simple to operate, with low cost, and can shorten maturing stage of dendrobium candidum fruit widely, efficient and do not have a damage by disease and insect, can the anniversary produce the dendrobium candidum seed, the transplanting survival rate of Cheng Miao can reach more than 95%, thereby opens up a new way for the breeding of seedlings of Dendrobium officinale.
Specific implementation method
Following examples are to further specify of the present invention, are not limitations of the present invention.That uses among the embodiment spends precious No. 1 (HYPONeX 1) and spends precious No. 2 (HYPONeX 2) to be Taiwan produced in USA platform and gardening enterprise stock action Co., Ltd packing product.
Embodiment 1:
The maternal plant of choosing robust growth when blooming carries out artificial pollination, and the back 120 days fruit maturations of pollinating are used for sowing with fruit as explant.During sowing, explant is used successively volume fraction 70% alcohol-pickled 30 seconds, sterilization is 20 minutes in the mercuric chloride solution of mass fraction 0.1%, rinsed with sterile water 4 times, cut fruit then, (every liter contains and spends precious No. 1 1.5g in the medium with transfer needle the Powdered embryo of yellow-white to be inoculated into the seed germination medium, peptone 0.5g, coconut milk 50mL, sucrose 15g, agar 6g, active carbon 1g, all the other are vitamin and inositol composition in the MS medium) on, seed germination after 10 days, form protocorm, form seedling after 80 days.
(every liter contains 6-benzyl purine (6-BA) 0.5mg to the seedling 100 strain rolling bottles that 2～3cm is high to the test tube flowering medium, methyl (NAA) 0.1mg, all the other are the MS medium) on, 20 strains are bloomed in the time of 45 days, flowering rate can reach 20%, choosing in vitro, the plant of robust growth carries out artificial pollination in vitro, 55 days fruit maturations, adopt down fruit, fruit need not sterilization, cuts fruit and carries out aseptic seeding, and left and right sides seed germination formed protocorm in 7 days, can form complete plantlet in the time of 80 days, the quantity of each fruit is more than 50,000 strains.
The intensive plantlet that aseptic seeding is obtained is transferred to the strong seedling culture base after separately, and (every liter contains and spends precious No. 1 1.5g, spends precious No. 2 1.5g, peptone 0.5g, caseinhydrolysate 0.1g, bananas juice 50g, carrot juice 10g, apple juice 10g, sucrose 10g, agar 6g, active carbon 1g, NAA 0.5mg, all the other are vitamin and inositol composition in the MS medium) go up and cultivate, 80 days formation plant height 3～4cm, every clump of Cheng Miao that 2～3 buds are arranged.
To become seedling to transfer to the natural daylight lower refining seedling 10 days, illuminance 1500～20001x, illumination 12 hours/day, then it is taken out from vial, clean the medium of root, in sphagna and the Lan Shi matrix that 1:1 mixes by volume, cultivate, keep suitably ventilating and enough humidity, 1000 strains survive 950 strains, and the survival rate of transplanting all can reach 95%.
Used medium is pH5.2～5.4 in above-mentioned experiment, cultivation temperature (26 ± 2) ℃.
Embodiment 2:
The maternal plant of choosing robust growth when blooming carries out artificial pollination, and the back 110 days fruit maturations of pollinating are used for sowing with fruit as explant.During sowing, explant is used alcohol-pickled 40 seconds of volume fraction 75% successively, and sterilization is 30 minutes in the mercuric chloride solution of mass fraction 0.15%, rinsed with sterile water 5 times, cut fruit then, (every liter contains and spends precious No. 1 1g, peptone 1g, coconut milk 80mL in the medium with transfer needle the Powdered embryo of yellow-white to be inoculated into the seed germination medium, sucrose 25g, agar 6.5g, active carbon 0.8g, all the other are vitamin and inositol composition in the MS medium) on, seed germination after 10 days, form protocorm,, protocorm is formed the line and staff control of bud and protocorms in order to obtain more seedling in process of production, (every liter contains and spends precious No. 1 1g at the shoot proliferation medium, peptone 0.5g, coconut milk 50mL, sucrose 10g, agar 6g, active carbon 1g, 6-benzyl purine 0.5mg, all the other are vitamin and inositol composition in the MS medium) last shoot proliferation, propagation is 4 times after 40 days, the protocorms that forms is proceeded propagation, obtains the seedling with root, and the quantity of each fruit is more than 50,000 strains.
With seedling 100 rolling bottles of 2～3cm high-band root to test tube flowering medium (every liter contains 6-BA 1.0mg, NAA 0.2mg, all the other are the MS medium), 22 strains are bloomed in the time of 45 days, flowering rate can reach 22%, chooses in vitro that the plant of robust growth carries out artificial pollination in vitro, 50 days fruit maturations, adopt down fruit, fruit need not sterilization, cuts fruit and carries out aseptic seeding, and left and right sides seed germination formed protocorm in 7 days, can form complete plantlet in the time of 80 days, the quantity of each fruit is more than 50,000 strains.
The intensive plantlet that aseptic seeding is obtained is transferred to the strong seedling culture base after separately, and (every liter contains and spends precious No. 1 1g, spends precious No. 2 1g, peptone 2g, caseinhydrolysate 0.3g, bananas juice 150g, carrot juice 30g, apple juice 30g, sucrose 20g, agar 6.5g, active carbon 2g, NAA 1.0mg, all the other are vitamin and inositol composition in the MS medium) go up and cultivate, 70 days formation plant height 3～4cm, every clump of Cheng Miao that 2～3 buds are arranged.
To become seedling to transfer to the natural daylight lower refining seedling 8 days, illuminance 1500～20001x, illumination 14 hours/day, then it is taken out from vial, clean the medium of root, in sphagna and the Lan Shi matrix that 1:1 mixes by volume, cultivate, keep suitably ventilating and enough humidity, 1000 strains survive 965 strains, and the survival rate of transplanting all can reach 96.5%.
Used medium is pH5.2～5.4 in above-mentioned experiment, cultivation temperature (26 ± 2) ℃.
Embodiment 3:
The maternal plant of choosing robust growth when blooming carries out artificial pollination, and the back 130 days fruit maturations of pollinating are used for sowing with fruit as explant.During sowing, explant is used alcohol-pickled 60 seconds of volume fraction 80% successively, and sterilization is 30 minutes in the mercuric chloride solution of mass fraction 0.2%, rinsed with sterile water 4 times, cut fruit then, (every liter contains and spends precious No. 1 1.5g, peptone 2g, coconut milk 100mL in the medium with transfer needle the Powdered embryo of yellow-white to be inoculated into the seed germination medium, sucrose 30g, agar 7g, active carbon 1g, all the other are vitamin and inositol composition in the MS medium) on, seed germination after 10 days, form protocorm,, protocorm is formed the line and staff control of bud and protocorms in order to obtain more seedling in process of production, (every liter contains and spends precious No. 1 2g at the shoot proliferation medium, peptone 2g, coconut milk 100mL, sucrose 20g, agar 7g, 6-benzyl purine 1.0mg, active carbon 2g, all the other are vitamin and inositol composition in the MS medium) last shoot proliferation, propagation is 4 times after 40 days, the protocorms that forms is proceeded propagation, obtains the seedling with root, and the quantity of each fruit is more than 50,000 strains.
The seedling 100 strain rolling bottles that 2～3cm is high are to test tube flowering medium (every liter contains 6-BA 0.5mg, NAA 0.1mg, all the other are the MS medium), 40 strains are bloomed in the time of 45 days, flowering rate can reach 40%, chooses in vitro that the plant of robust growth carries out artificial pollination in vitro, 60 days fruit maturations, adopt down fruit, fruit need not sterilization, cuts fruit and carries out aseptic seeding, and left and right sides seed germination formed protocorm in 7 days, can form complete plantlet in the time of 80 days, the quantity of each fruit is more than 50,000 strains.
The intensive plantlet that aseptic seeding is obtained is transferred to the strong seedling culture base after separately, and (every liter contains and spends precious No. 1 1.2g, spends precious No. 2 1.2g, peptone 1g, caseinhydrolysate 0.2g, bananas juice 100g, carrot juice 20g, apple juice 20g, sucrose 15g, agar 7g, active carbon 1.5g, NAA 0.8mg, all the other are vitamin and inositol composition in the MS medium) go up and cultivate, 60 days formation plant height 3～4cm, every clump of Cheng Miao that 2～3 buds are arranged.
To become seedling to transfer to the natural daylight lower refining seedling 12 days, illuminance 1500～20001x, illumination 16 hours/day, then it is taken out from vial, clean the medium of root, in peat and the Lan Shi matrix that 1:1 mixes by volume, cultivate, keep suitably ventilating and enough humidity, 1000 strains survive 980 strains, and the survival rate of transplanting all can reach 98%.
Used medium is pH5.2～5.4 in above-mentioned experiment, cultivation temperature (26 ± 2) ℃.
Claims (3)
1. seedlings of Dendrobium officinale propagation method, its feature comprises the steps:
Dendrobium candidum (the Dendrobium officinale Kimura et Migo) maternal plant of choosing robust growth when (1) blooming carries out artificial pollination, pollination back fruit maturation, with fruit as explant;
(2) explant is used successively alcohol-pickled, mercuric chloride solution sterilization is cut after the rinsed with sterile water, Powdered embryo is inoculated into the seed germination medium, seed germination forms protocorm, and further forms seedling, and described seed germination medium contains for every liter spends precious No. 1 1～1.5g, peptone 0.5～2g, coconut milk 50～100mL, sucrose 15～30g, agar 6～7g, active carbon 0.5～1g, all the other are vitamin and inositol composition in the MS medium;
(3) seedling is used the test tube flowering medium culture, the plant of choosing robust growth after blooming carries out artificial pollination in vitro, cut behind the fruit maturation and carry out aseptic seeding, seed germination forms protocorm, and further form complete plantlet, described test tube flowering medium is every liter and contains 6-benzyl purine 0.5～1.0mg, methyl 0.1～0.2mg, and all the other are the MS medium;
(4) plantlet is transplanted on the strong seedling culture base is cultivated, be formed into seedling, described strong seedling culture base contains for every liter spends precious No. 1 1～1.5g, spend precious No. 2 1～1.5g, peptone 0.5～2g, caseinhydrolysate 0.1～0.3g, bananas juice 50～150g, carrot juice 10～30g, apple juice 10～30g, sucrose 10～20g, agar 6～7g, active carbon 1～2g, methyl 0.5～1.0mg, all the other are vitamin and inositol composition in the MS medium;
(5) will become seedling at the natural daylight lower refining seedling, clean the medium of root then, in matrix, cultivate, obtain seedling with sphagna or peat and Lan Shi mixing in 1: 1 by volume;
Medium pH 5.2～5.4 in above-mentioned steps (2)～(4), cultivation temperature (26 ± 2) ℃.
2. a kind of seedlings of Dendrobium officinale propagation method according to claim 1, the described alcohol concentration of step (2) is a volume fraction 70%～80%, soak time preferably 30～60 seconds, the concentration of described mercuric chloride solution is mass fraction 0.1%～0.2% preferably, disinfecting time preferably 20～30 minutes, the rinsing number of times of described sterile water is 4～5 times, the height of the described seedling of step (3) is 2～3cm, the described one-tenth height of seedling of step (4) 3～4cm, every clump has 2～3 buds, the described hardening time of step (5) is 8～12 days, and the illumination of described natural lighting is 1500～20001x, and light application time is 12～16 hours/day.
3. a kind of high-efficient propagation method of seedlings of Dendrobium officinale according to claim 1 and 2, it is characterized in that the described protocorm of step (2) is formed the line and staff control of bud and protocorms, on the shoot proliferation medium, carry out shoot proliferation, the protocorms that forms is proceeded propagation, the seedling of band root then enters the described cultivation of step (3), described shoot proliferation medium contains for every liter spends precious No. 1 1～2g, peptone 0.5～2g, coconut milk 50～100mL, sucrose 10～20g, agar 6～7g, 6-benzyl purine 0.5～1.0mg, active carbon 1～2g, all the other are vitamin and inositol composition in the MS medium.
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