CN103814821A - Method for efficiently and quickly producing bletilla striata seedling - Google Patents
Method for efficiently and quickly producing bletilla striata seedling Download PDFInfo
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Abstract
The invention aims at providing a method for efficiently and quickly producing a bletilla striata seedling. The method comprises (1) a liquid culture stage and (2) a solid medium culture stage, wherein (1) the liquid culture stage comprises the particular steps that a bletilla striata capsule is disinfected via sodium hypochlorite; the capsule is cut; a seed is sown in a liquid medium for culture; the seed is developed into a protocorm; (2) the solid medium culture stage comprises the particular steps that the protocorm is induced to be differentiated into a root-free seedling, and then transferred to a rooting culture medium for rooting culture, and a cultivation substrate transplanted into a greenhouse is domesticated before field transplanting. The method realizes efficient and quick production of the bletilla striata seedling; compared with whole-course solid culture, the method overcomes the defect that the seed germination rate is low when an early seed is cultured on a solid medium; the seed germination rate is increased significantly and reaches 99%; the fresh weight and the dry weight of the protocorm are increased greatly; and the whole production cycle is shortened significantly, and can be shortened to about 110 days.
Description
Technical field
The invention belongs to micropropagation of plants technical field, be specifically related to a kind of efficient method of producing fast bletilla striata seedling.
Background technology
The bletilla striata (
bletilla Striata(Thunb.) Reichb.f.) for the bletilla striata belongs to herbaceos perennial, be national key protected wild plants, not only ornamental value is higher, is also one of conventional Chinese medicine.The bletilla striata is used as medicine with dry tuber, has astringing to arrest bleeding, effect of detumescence and promoting granulation, to spitting of blood, spit blood, have blood in stool and trauma hemostasis, sore swollen toxin, brothers cross mountains to split etc. good therapeutic effect, be widely used in treatment pulmonary tuberculosis and bronchiectasis hemoptysis, the diseases such as ulcer bleeding, the main subject of a sale of the bletilla striata has hospital, biopharmaceutical company, medicinal material dealer, chemical plant etc., its purposes is not only embodied in conventional stomach medicine miscellaneous in people's life, stitching agent in medical operating, traumatism and bleeding, burn and scald, brothers are broken etc. in the treatment of disease, also be embodied in ornamental plantation application, women's skin protection cosmetics, the senior cigarette stub glue that the male sex smokes, industrial thickener, slurry silk, sizing or do in raw material and the wine brewing etc. such as coating.
In recent years, along with the continuous soaring market demand, wild bletilla striata degree of meeting with is excavated, and wild bletilla striata resource is endangered.Solving bletilla striata seedling quick propagating technology is the key of alleviating the demand and supply contraction.And the common whole production process of existing quick reproduction technique has taked planting seed to solid culture medium, and then through stages such as breaking up, take root, lead the problems such as seed germination rate is low, protocorm growth retardation, production cycle length.
Summary of the invention
The invention provides a kind of efficient method of producing fast bletilla striata seedling, its objective is by the liquid oscilaltion cultivation of first stage and the solid culture of second stage, in the hope of overcoming some drawbacks of omnidistance solid culture, improve bletilla striata seeds germination rate, promote fresh weight and the dry weight of protocorm, accelerate protocorm growth rate, thereby shorten the production cycle.It is first bletilla striata seeds to be placed in to liquid nutrient medium to cultivate, and after seed development becomes protocorm, then induces seedling differentiation, and then moves to green house domestication.
Employing the invention has the beneficial effects as follows early stage cultivation stage fully take into account bletilla striata seeds sprout, protocorm growth characteristic, adopt liquid culture, make seed fully contact nutriment with protocorm, accelerated seed germination and protocorm growth rate; Simultaneously also fully take into account differentiation, take root, nutrition and the growth characteristic of the developmental stage such as domestication, medium and domestication matrix rational formula, domestication matrix fertilizer conservation, water conservation, good permeability, porous has greatly promoted that the imaginary root of plant is g and D, has fabulous fixation to bletilla striata seedling; Finally realized bletilla seed height of seedling effect and produced fast, seed germination rate significantly improves, and reaches 99%, and protocorm grows rapidly, and the whole production cycle can foreshorten to about 110 days.
A kind of efficient method of producing fast bletilla striata seedling provided by the invention comprises the following steps.
Bletilla striata capsule is first used to alcohol-pickled 30～50 seconds, then be positioned in the aqueous sodium hypochlorite solution that effective chlorine density is 0.3-0.8% and soak 10～15 minutes, then use rinsed with sterile water 3～4 times.
Capsule after sterilization is cut, bletilla striata seeds is sowed in liquid nutrient medium to shaken cultivation; Liquid nutrient medium consists of: 1/4MS medium+6-benzyl purine 0.3-0.8 mg/litre+methyl α-naphthyl acetate 2.5-3.2 mg/litre+murphy juice 120-150 grams per liter+sucrose 25-30 grams per liter+pH 5.8-6.3; Liquid culture condition is, under 18～24 degrees Celsius, 180～230 revs/min of rotating speeds, illuminance 1800～2300 luxs, the light application time condition of 4～6 hours/day, and shaken cultivation 15～20 days, seed germination forms protocorm.
The protocorm obtaining is placed in to differential medium, under 25～28 degrees Celsius, illuminance 2000～2500 luxs, the light application time condition of 10～12 hours/day, cultivates 30～40 days, be first divided into budlet, then become the unrooted seedling with 3 leaves; Differential medium consists of: MS medium+methyl α-naphthyl acetate 0.5～0.8 mg/litre+6-benzyl purine 1.3～1.5 mg/litre+bananas juice, 100～110 grams per liters+sucrose, 25～3 0 grams per liters+agar 6～7 grams per liters+pH5.6～5.8.
The seedling obtaining is transferred in root media, and temperature is controlled at 24～28 degrees Celsius, cultivates after 30～40 days, obtains 2～3.5 centimetres of high seedlings; Wherein, described root media consists of: MS medium+methyl α-naphthyl acetate 2.5～3 mg/litre+sucrose 25～30 grams per liters+active carbon 0.5～0.8 mg/litre+agar 7-8 grams per liter+pH 6～6.2.
The seedling obtaining is taken out in bottle with clear water and rinse and remove medium, with carbendazim solution immersion 30～60 minutes, be transplanted in the cultivation matrix in green house; Cultivation matrix is by volume (0.4～0.7) (1.5～2) of perlite, vermiculite and turfy soil: the mixture of (2.5～3) composition; Wherein perlite particle diameter 4～7 mm, the particle diameter of vermiculite is 3～4 mm, the content of organics of turfy soil is 30%～40%; Cultivate about 20 days, be the seedling of production.
The present invention has finally realized bletilla seed height of seedling effect and has produced fast, and seed germination rate significantly improves, and reaches 99%, and protocorm grows rapidly, and the whole production cycle can foreshorten to about 110 days.
Accompanying drawing explanation
Fig. 1: the sprouting situation in bletilla striata seeds liquid medium within.
Fig. 2: cultivate the bletilla striata protocorm (wherein scheming ABC is the protocorm that liquid nutrient medium is cultivated, and figure D is the protocorm that solid culture medium is cultivated) obtaining.
Fig. 3: the bletilla striata of solid culture medium cultivation stage.
Fig. 4: the bletilla striata has just been transplanted to the growing state (wherein scheming ABC is the bletilla striata seedling in liquid nutrient medium source, and figure D is the bletilla striata seedling in solid culture medium source) after the dish of cave.
Fig. 5: the seedling of production.
Embodiment
The feature that the present invention is outstanding and marked improvement can be embodied from following example, but can not impose any restrictions the present invention.
Embodiment 1:
Bletilla striata capsule is first used to alcohol-pickled 35 seconds, then to be positioned over effective chlorine density be to soak 10 minutes in 0.8% aqueous sodium hypochlorite solution, then use rinsed with sterile water 3～4 times.
Capsule after sterilization is cut, bletilla striata seeds is sowed in liquid nutrient medium to shaken cultivation; Growing state after seed germination is shown in Figure 1A; Simultaneously also by seed broadcasting in solid culture medium, experimental group in contrast; Liquid nutrient medium consists of: 1/4MS medium+6-benzyl purine 0.6 mg/litre+methyl α-naphthyl acetate 3.1 mg/litre+murphy juice 150 grams per liters+sucrose 30 grams per liters+pH 6.2; Solid culture medium forms to add outside agar 7 grams per liters, the same liquid nutrient medium of other composition; Liquid culture condition is, under 19 degrees Celsius, 220 revs/min of rotating speeds, illuminance 2200 luxs, the light application time condition of 6 hours/day, and shaken cultivation 19 days, seed germination forms protocorm, see Fig. 2 A, 500 protocorm fresh weights are 4.243 grams, and dry weight is 0.421 gram; Solid culture medium condition of culture is: under 19 degrees Celsius, illuminance 2200 luxs, the light application time condition of 6 hours/day, cultivate 33 days, seed germination forms protocorm, sees Fig. 2 D, and 500 protocorm fresh weights are 0.924 gram, and dry weight is 0.109 gram.
The protocorm obtaining is placed in to differential medium, under 28 degrees Celsius, illuminance 2500 luxs, the light application time condition of 11 hours/day, cultivate 32 days, first be divided into budlet, become again the unrooted seedling with 3 leaves, and under condition of culture similarly, the protocorm that solid culture medium is cultivated out grows up to the unrooted seedling of 3 leaves need about 42 days; Differential medium consists of: MS medium+methyl α-naphthyl acetate 0.8 mg/litre+6-benzyl purine 1.4 mg/litre+bananas juice 100～110 grams per liters+sucrose 30 grams per liters+agar 7 grams per liters+pH5.8.
The seedling obtaining is transferred in root media, and temperature is controlled at 27 degrees Celsius, cultivates after 35 days, obtains 2～3.5 centimetres of high seedlings; And under condition of culture similarly, the seedling in solid culture medium source grows up to the seedling needs 45 days of taking root; Wherein, described root media consists of: MS medium+methyl α-naphthyl acetate 2.8 mg/litre+sucrose 30 grams per liters+active carbon 0.8 mg/litre+agar 8 grams per liters+pH 6.2.
The seedling obtaining is taken out in bottle with clear water and rinse and remove medium, with carbendazim solution immersion 50 minutes, be transplanted in the cultivation matrix in green house, see Fig. 4; Cultivation matrix is the by volume mixture of 0.6::2:2.8 composition of perlite, vermiculite and turfy soil; Wherein perlite particle diameter 4～6 mm, the particle diameter of vermiculite is 3～4 mm, the content of organics of turfy soil is 38%; Cultivate about 20 days, be the seedling of production; And under condition of culture similarly, the bletilla striata seedling domestication in solid culture medium source needs 30 left and right.
Embodiment 2:
Bletilla striata capsule is first used to alcohol-pickled 35 seconds, then be positioned in 0.5% aqueous sodium hypochlorite solution and soak 15 minutes, then use rinsed with sterile water 3～4 times.
Capsule after sterilization is cut, bletilla striata seeds is sowed in liquid nutrient medium to shaken cultivation; Growing state after seed germination is shown in Figure 1B; Liquid nutrient medium consists of: 1/4MS medium+6-benzyl purine 0.3 mg/litre+methyl α-naphthyl acetate 2.5 mg/litre+murphy juice 140 grams per liters+sucrose 25 grams per liters+pH 6.0; Liquid culture condition is, under 18 degrees Celsius, 180 revs/min of rotating speeds, illuminance 1800 luxs, the light application time condition of 4 hours/day, and shaken cultivation 15 days, seed germination forms protocorm, see Fig. 2 B, 500 protocorm fresh weights are 2.358 grams, and dry weight is 0.246 gram.
The protocorm obtaining is placed in to differential medium, under 25 degrees Celsius, illuminance 2200 luxs, the light application time condition of 10～12 hours/day, cultivates 35 days, be first divided into budlet, then become the unrooted seedling with 3 leaves; Differential medium consists of: MS medium+methyl α-naphthyl acetate 0.5 mg/litre+6-benzyl purine 1.3 mg/litre+bananas juice 100～110 grams per liters+sucrose 28 grams per liters+agar 6 grams per liters+pH5.6.
The seedling obtaining is transferred in root media, and temperature is controlled at 24 degrees Celsius, cultivates after 38 days, obtains 2～3.5 centimetres of high seedlings, sees Fig. 3; Wherein, described root media consists of: MS medium+methyl α-naphthyl acetate 2.6 mg/litre+sucrose 25 grams per liters+active carbon 0.6 mg/litre+agar 7 grams per liters+pH 6.
The seedling obtaining is taken out in bottle with clear water and rinse and remove medium, with carbendazim solution immersion 35 minutes, be transplanted in the cultivation matrix in green house; Cultivation matrix is the by volume mixture of 0.7:2:3 composition of perlite, vermiculite and turfy soil; Wherein perlite particle diameter 5～7 mm, the particle diameter of vermiculite is 3～4 mm, the content of organics of turfy soil is 30%; Cultivate about 20 days, be the seedling of production.
Embodiment 3:
Bletilla striata capsule is first used to alcohol-pickled 50 seconds, then to be positioned over effective chlorine density be to soak 12 minutes in 0.6% aqueous sodium hypochlorite solution, then use rinsed with sterile water 3～4 times.
Capsule after sterilization is cut, bletilla striata seeds is sowed in liquid nutrient medium to shaken cultivation; Growing state after seed germination is shown in Fig. 1 C; Liquid nutrient medium consists of: 1/4MS medium+6-benzyl purine 0.8 mg/litre+methyl α-naphthyl acetate 2.8 mg/litre+murphy juice 150 grams per liters+sucrose 25 grams per liters+pH 5.8; Liquid culture condition is, under 18 degrees Celsius, 230 revs/min of rotating speeds, illuminance 2200 luxs, the light application time condition of 4～5 hours/day, and shaken cultivation 20 days, seed germination forms protocorm, see Fig. 2 C, 500 protocorm fresh weights are 2.246 grams, and dry weight is 0.231 gram.
The protocorm obtaining is placed in to differential medium, under 28 degrees Celsius, illuminance 2000 luxs, the light application time condition of 10～12 hours/day, cultivates 38 days, be first divided into budlet, then become the unrooted seedling with 3 leaves; Differential medium consists of: MS medium+methyl α-naphthyl acetate 0.8 mg/litre+6-benzyl purine 1.5 mg/litre+bananas juice 110 grams per liters+sucrose 30 grams per liters+agar 7 grams per liters+pH5.8.
The seedling obtaining is transferred in root media, and temperature is controlled at 27 degrees Celsius, cultivates after 35 days, obtains 2～3.5 centimetres of high seedlings; Wherein, described root media consists of: MS medium+methyl α-naphthyl acetate 3 mg/litre+sucrose 30 grams per liters+active carbon 0.7 mg/litre+agar 8 grams per liters+pH 6.
The seedling obtaining is taken out in bottle with clear water and rinse and remove medium, with carbendazim solution immersion 50 minutes, be transplanted in the cultivation matrix in green house; Cultivation matrix is the by volume mixture of 0.5:1.8:2.8 composition of perlite, vermiculite and turfy soil; Wherein perlite particle diameter 5～7 mm, the particle diameter of vermiculite is 3～4 mm, the content of organics of turfy soil is 40%; Cultivate about 20 days, be the seedling of production, see Fig. 5.
Claims (5)
1. an efficient method of producing fast bletilla striata seedling, its feature comprises the following steps:
The first cultivation stage,
(1) bletilla striata capsule is first used to alcohol-pickled 30～50 seconds, then be positioned in aqueous sodium hypochlorite solution and soak 10～15 minutes, then use rinsed with sterile water 3～4 times, the capsule after sterilization is cut, get seed for subsequent use;
(2) bletilla striata seeds step (1) being obtained is sowed in liquid nutrient medium, shaken cultivation, and seed germination forms protocorm, for subsequent use;
The second cultivation stage,
(3) protocorm step (2) being obtained is placed in differential medium, under 25～28 degrees Celsius, illuminance 2000～2500 luxs, the light application time condition of 10～12 hours/day, cultivate 30～40 days, first be divided into budlet, then become the unrooted seedling with 3 leaves; Wherein, described differential medium consists of: MS medium+methyl α-naphthyl acetate 0.5～0.8 mg/litre+6-benzyl purine 1.3～1.5 mg/litre+bananas juice, 100～110 grams per liters+sucrose, 25～3 0 grams per liters+agar 6～7 grams per liters+pH5.6～5.8;
(4) seedling obtaining in step (3) is transferred in root media, temperature is controlled at 24～28 degrees Celsius, cultivates after 30～40 days, obtains 2～3.5 centimetres of high seedlings; Wherein, described root media consists of: MS medium+methyl α-naphthyl acetate 2.5～3 mg/litre+sucrose 25～30 grams per liters+active carbon 0.5～0.8 mg/litre+agar 7-8 grams per liter+pH 6～6.2;
(5) seedling step (4) being obtained is taken out with clear water and is rinsed and remove medium in bottle, with carbendazim solution immersion 30～60 minutes, is transplanted in the cultivation matrix in green house, cultivates about 20 days, is the seedling of production; Described cultivation matrix is by volume (0.4～0.7) (1.5～2) of perlite, vermiculite and turfy soil: the mixture of (2.5～3) composition.
2. efficient method of producing fast bletilla striata seedling according to claim 1, is characterized in that, in the first cultivation stage step (1), in described aqueous sodium hypochlorite solution, effective chlorine density is 0.3-0.8%.
3. efficient method of producing fast bletilla striata seedling according to claim 1, it is characterized in that, the first liquid culture condition of cultivating step rapid (2) is, under 18～24 degrees Celsius, 180～230 revs/min of rotating speeds, illuminance 1800～2300 luxs, the light application time condition of 4～6 hours/day, shaken cultivation 15～20 days, seed germination forms protocorm.
4. the efficient method of producing fast bletilla striata seedling of scale according to claim 1; it is characterized in that; first cultivates in step rapid (2), and described liquid nutrient medium consists of: 1/4MS medium+6-benzyl purine 0.3-0.8 mg/litre+methyl α-naphthyl acetate 2.5-3.2 mg/litre+murphy juice 120-150 grams per liter+sucrose 25-30 grams per liter+pH 5.8-6.3.
5. the efficient method of producing fast bletilla striata seedling of scale according to claim 1; it is characterized in that, in the second cultivation stage step (5), matrix formulations is perlite particle diameter 4～7 mm; the particle diameter of vermiculite is 3～4 mm, and the content of organics of turfy soil is 30%～40%.
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| CN104604680A (en) * | 2015-01-19 | 2015-05-13 | 杭州雷威农业开发有限公司 | Growth medium capable of promoting seed germination and seedling growth of bletilla striata, formula of growth medium and preparation method |
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| CN105325302A (en) * | 2015-12-11 | 2016-02-17 | 电子科技大学 | Method for bletilla striata seedling production based on liquid medium |
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| CN104137778B (en) * | 2014-07-30 | 2016-05-04 | 中国科学院南京分院东台滩涂研究院 | A kind of bletilla seed liquid suspension is cultivated the method for protocorm |
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| CN104604680A (en) * | 2015-01-19 | 2015-05-13 | 杭州雷威农业开发有限公司 | Growth medium capable of promoting seed germination and seedling growth of bletilla striata, formula of growth medium and preparation method |
| CN104737911A (en) * | 2015-03-30 | 2015-07-01 | 云南中医学院 | Quick cultivation method for rhizoma bletillae tissue culture seedlings |
| CN104871977A (en) * | 2015-05-29 | 2015-09-02 | 江苏农林职业技术学院 | Bletilla striata tissue culture medium and its preparing method |
| CN105075858A (en) * | 2015-05-29 | 2015-11-25 | 浙江中医药大学 | Liquid rapid-propagation method for rhizoma bletillae seeds |
| CN105052752A (en) * | 2015-09-17 | 2015-11-18 | 康美药业（文山）药材种植管理有限公司 | Culture medium for bletilla striata tissue culture |
| CN105340740A (en) * | 2015-11-10 | 2016-02-24 | 云南师范大学 | Liquid high-efficiency germination induction solution and rapid propagation method for bletilla striata seeds |
| CN105325302A (en) * | 2015-12-11 | 2016-02-17 | 电子科技大学 | Method for bletilla striata seedling production based on liquid medium |
| CN105766459A (en) * | 2016-03-16 | 2016-07-20 | 陕西理工学院 | Simple and convenient method for rhizome-bletillae-protocorm seedling culturing with biodegradable membranous material |
| CN105766321A (en) * | 2016-03-22 | 2016-07-20 | 镇远县道益堂生物科技有限公司 | Sexual propagation seedling culture method for Bletilla striata |
| CN105815068A (en) * | 2016-03-22 | 2016-08-03 | 镇远县道益堂生物科技有限公司 | Method for producing bletilla striata seed stems |
| CN106416972A (en) * | 2016-08-29 | 2017-02-22 | 浙江金石生物科技有限公司 | Method for quickly cultivating bletilla striata seedlings |
| CN109076923A (en) * | 2018-08-28 | 2018-12-25 | 河南云帮农业科技有限公司 | A kind of bletilla striata aseptic seeding special culture media |
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Application publication date: 20140528 |