CN103583365B - Production method of bletilla seed stems - Google Patents
Production method of bletilla seed stems Download PDFInfo
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- CN103583365B CN103583365B CN201310579744.9A CN201310579744A CN103583365B CN 103583365 B CN103583365 B CN 103583365B CN 201310579744 A CN201310579744 A CN 201310579744A CN 103583365 B CN103583365 B CN 103583365B
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Abstract
The invention discloses a production method of bletilla seed stems, which comprises the following steps: 1) explant treatment and inoculating, 2) protocorm obtaining and multiplication, 3) differentiation and seedling forming of protocorms, 4) test-tube corm obtaining, 5) primary seed stem obtaining and 6) secondary seed stem obtaining. According to the method, mature bletilla seeds are subjected to aseptic seeding and culture, many test-tube seed stems are obtained, and the larger production seed stems capable of being grown in a large field are obtained by primary and secondary seed stem reproduction. The method solves the problem of a low survival rate of direct exercising of tissue culture seedlings, provides a seed source for manual massive bletilla planting, and lays a foundation for sustainable development, exploitation and utilization of bletilla.
Description
Technical field
The present invention relates to biological technical field, especially a kind of production method of bletilla seed stem.
Background technology
The bletilla striata " Bletilla striata(Thunb.) Reichb.f.) belong to herbaceos perennial for the orchid family bletilla striata; call white root, Cold boiled chicken baby, connect splendid achnatherum grass, purple blue etc.; be a kind of conventional bulk medicinal materials; be used as medicine with dry tuber; its cold nature, bitter, sweet, puckery, return lung, stomach, Liver Channel; there is the functions such as tonifying lung hemostasis, detumescence and promoting granulation, the disease such as cure mainly pulmonary tuberculosis hemoptysis, bronchiectasis hemoptysis, stomach ulcer haematemesis, hematuria, have blood in stool; The diseases such as traumatism and bleeding, burn and scald, rhagadia manus et pedis are controlled in external application; The another bletilla striata, containing higher Bletilla glucomannan, can be used as supensoid agent and emulsifier, extensive use in food and chemical field; And be also widely used in biological medicine, health food, beauty culture, textile printing and dyeing, sapecial coating and daily-use chemical industry etc.Bletilla striata pattern is gorgeous, can do beautiful ornamental plants.Bletilla striata main product in Sichuan, Guizhou, Yunnan, Shaanxi, the province such as Gansu.
The bletilla striata is born in the hillside thick grass of high mountain and knob, the dark and damp place of sparse woods and mountain valley or cheuch rock seam, likes warm, shady and cool moistening environment; Leaf 2-6 piece, alternate, long and narrow round shape lanceolar to wire lanceolar, tool joint between blade and petiole, petiole is rolled up mutually and is embraced into bulbous, inflorescence top is raw, total shape, a normal tool number flower, usually branch or extremely rare branch, flower do not have aubergine, pink, yellow or white, sepal is similar to petal, closely isometric, from life; More than mesochil obvious 3 split, and side sliver is upright, pollen load 8, and become 2 groups, 4, every room, grain opaque, 1, column cap, is positioned under rostellum, eat the oval shape spindle of fruit, uprightly, and the fruit phase 7-9 month.This platymiscium has and expands underground stem tuber, in triangular shape oblate spheroid or regular rhombus, and its nearby normal many pieces of the previous years of tool and in the past annual remained pseudobulb.
For a long time, bletilla striata market mainly relies on wild resource.Due to extensive, the increase of demand and the surging of price of purposes, cause resource to be ransacked of formula and excavate, add the destruction of ecotope, wild resource is day by day exhausted, and the bletilla striata has become China second class protection plant.
Adopt artificial breeding to be the effective measure meeting bletilla striata market, but provenance become " bottleneck " of development.Bletilla striata seeds is seed propagation difficulty under field conditions (factors), and stem tuber is cut breeding and is easier to, but need the amount of kind large, reproduction coefficient is low, disease resistance ability, can degenerate year by year, is difficult to the needs meeting large-scale production.
Summary of the invention
The object of the invention is: the production method that a kind of bletilla seed stem is provided, which overcome the problem that bletilla striata plantlet in vitro direct hardening survival rate is low, solve the kind source problem of bletilla striata manual scale planting, for the sustainable development of the bletilla striata and exploitation are laid a good foundation.
The present invention is achieved in that the production method of bletilla seed stem, comprises the steps:
1) process of explant and inoculation: gather bletilla striata capsule in the 9-10 month, surface dirt is rinsed well with water, naturally after drying, superclean bench is alcohol-pickled sterilization 30 s of 75% with mass percent concentration, use 0.1% mercuric chloride liquid soaking disinfection 8 min again, then after using sterile water wash 5 times, blot with the moisture of aseptic filter paper by bletilla striata capsule surface again, after seed in bletilla striata capsule is taken out, broadcast sowing the surface of seed germination medium uniformly, cultivate in culturing room, cultivation temperature is 25 ± 2 DEG C, intensity of illumination is 1500-2000Lx, light application time is 12h/d, the main body of described germination medium is 1/2MS medium, and wherein NAA is 0.5mg/L, 6-BA is 1.0 mg/L, and the mass percent of white sugar is 3%, and the mass percent of agar powder is 0.7%, pH is 5.8,
2) acquisition of protocorm and propagation: seed culture is after 10 days, form yellow protocorm, be transferred to when protocorm does not differentiate blade in proliferated culture medium, be positioned over culturing room's cultivation that temperature is 25 ± 2 DEG C, intensity of illumination 1500-2000Lx, light application time 12h/d, cultivated after 25 days, formed shape protocorm of growing thickly; The main body of described proliferated culture medium is MS medium, and wherein NAA is 0.2mg/L, 6-BA is 0.5 mg/L, and the mass percent of white sugar is 3%, and the mass percent of agar powder is 0.7%, pH is 5.8;
3) protocorm differentiation seedling: shape protocorm of growing thickly is transferred in differential medium, puts the culturing room of 25 ± 2 DEG C, intensity of illumination 1500-2000Lx, light application time 12h/d CMC model, to cultivating the plant obtaining 2-3 sheet leaf; The main body of described differential medium is MS medium, and wherein NAA is 0.3mg/L, and the mass percent of white sugar is 3%, and the mass percent of agar powder is 0.7%, pH is 5.8;
4) acquisition of cormel in vitro: the plant that step 3) obtains is transferred in root media, the culturing room being 25 ± 2 DEG C in temperature cultivates, and intensity of illumination 1500-2000Lx, light application time 12h/d, cultivated after 45-60 days, obtains cormel in vitro; The main body of described root media is MS medium, and wherein NAA is 0.5mg/L, 6-BA is 0.1mg/L, and the mass percent of white sugar is 3%, and the mass percent of agar powder is 0.7%, pH is 5.8;
5)
onelevel plants the acquisition of stem: the blake bottle being formed with cormel in vitro is placed on ambient temperatare and puts 3 days, then sealed membrane hardening is opened 2 days, with tweezers, the cormel in vitro in bottle is pressed from both sides out, medium is rinsed out under running water, and clean out dead leaf and root, plant in the mixed-matrix of ready vermiculite and thin bark, the volume ratio of vermiculite and thin bark is 4:1, thin bark particle diameter is less than 1cm, water 1000 times of carbendazols of drenching, then conveniently manage, start every first quarter moon during sprouting pending and spray one time of nutrition liquid; Treat that vegetative period terminates, blade is withered and yellow to come off, the bletilla striata bulb of underground part of gathering, and namely obtains one-level kind bulb;
6) secondary kind stem: when temperature gos up, be planted in by the one-level kind stem of acquisition in the matrix of vermiculite, humus soil and the mixing of thin bark, the volume ratio of vermiculite, humus soil and thin bark is 3:1:1, and thin bark particle diameter is less than 1cm, to drench 1000 times of carbendazols, conveniently manage; When kind of a stem sends sprouting, start every first quarter moon spray one time of nutrition liquid, until blade is withered and yellow come off time, underground part bletilla striata bulb of gathering, obtains and produces with planting the secondary kind stem of stem as cultivation.
When planting season, secondary kind stem can directly be planted in land for growing field crops, thus obtains commodity bulb.
Owing to have employed technique scheme; compared with prior art; the present invention utilizes bletilla striata mature seed to carry out aseptic seeding cultivation; obtain a large amount of test tube kind stems; again by the breeding of one-level, secondary kind stem, thus obtain larger production kind of the stem can planted in land for growing field crops, the method overcomes the low problem of plantlet in vitro direct hardening survival rate; solve the kind source problem of bletilla striata manual scale planting, for the sustainable development of the bletilla striata and exploitation are laid a good foundation.
Embodiment
Embodiments of the invention: the production method of bletilla seed stem, comprises the steps:
1) process of explant and inoculation: carry out artificial pollination at annual bletilla striata season of flowers, bletilla striata capsule is gathered in the 9-10 month, surface dirt is rinsed well with water, naturally after drying, superclean bench is alcohol-pickled sterilization 30 s of 75% with mass percent concentration, use 0.1% mercuric chloride liquid soaking disinfection 8 min again, then after using sterile water wash 5 times, blot with the moisture of aseptic filter paper by bletilla striata capsule surface again, longitudinally capsule is cut open with sterile scalpel, after seed in bletilla striata capsule is taken out, broadcast sowing the surface of seed germination medium uniformly, cultivate in culturing room, cultivation temperature is 25 ± 2 DEG C, intensity of illumination is 1500-2000Lx, light application time is 12h/d, the main body of described germination medium is 1/2MS medium, and wherein NAA is 0.5mg/L, 6-BA is 1.0 mg/L, and the mass percent of white sugar is 3%, and the mass percent of agar powder is 0.7%, pH is 5.8,
2) acquisition of protocorm and propagation: seed culture is after 10 days, form yellow protocorm, after the protocorm differentiation that has go out green leaf primordium, be transferred to when protocorm does not differentiate blade in proliferated culture medium, being positioned over temperature is that in the culturing room of 25 ± 2 DEG C, normal temperature is cultivated, intensity of illumination 1500-2000Lx, light application time 12h/d, cultivated after 25 days, formed shape protocorm of growing thickly, its proliferation times can reach 5-8 doubly, and it is green that part protocorm starts to produce dissociation; The main body of described proliferated culture medium is MS medium, and wherein NAA is 0.2mg/L, 6-BA is 0.5 mg/L, and the mass percent of white sugar is 3%, and the mass percent of agar powder is 0.7%, pH is 5.8;
3) protocorm differentiation seedling: shape protocorm of growing thickly is transferred in differential medium, put the culturing room of 25 ± 2 DEG C, intensity of illumination 1500-2000Lx, light application time 12h/d CMC model, about 10 days protocorms start to turn green, after differentiate leaf primordium and blade gradually, within 30 days, major part grows up to the plant of 2-3 sheet leaf; The main body of described differential medium is MS medium, and wherein NAA is 0.3mg/L, and the mass percent of white sugar is 3%, and the mass percent of agar powder is 0.7%, pH is 5.8;
4) acquisition of cormel in vitro: the plant that step 3) obtains is transferred in root media, the culturing room being 25 ± 2 DEG C in temperature cultivates, intensity of illumination 1500-2000Lx, light application time 12h/d, cultivate and start to form root for 15 days, rooting rate 100%, blade is grown up, and seedling base portion expands gradually, forms strong sprout, cultivate 45-60 days seedling leafs turn yellow withered fall, obtain cormel in vitro; The main body of described root media is MS medium, and wherein NAA is 0.5mg/L, 6-BA is 0.1mg/L, and the mass percent of white sugar is 3%, and the mass percent of agar powder is 0.7%, pH is 5.8;
5)
onelevel plants the acquisition of stem: the blake bottle being formed with cormel in vitro is placed on ambient temperatare and puts 3 days, then sealed membrane hardening is opened 2 days, with tweezers, the cormel in vitro in bottle is pressed from both sides out, medium is rinsed out under running water, and clean out dead leaf and root, plant in the mixed-matrix of ready vermiculite and thin bark, the volume ratio of vermiculite and thin bark is 4:1, thin bark particle diameter is less than 1cm, water 1000 times of carbendazols of drenching, then conveniently manage, start every first quarter moon during sprouting pending and spray one time of nutrition liquid, bulb germination survival rate more than 85%; Treat that vegetative period terminates, blade is withered and yellow to come off, the bletilla striata bulb of underground part of gathering, and namely obtains one-level kind bulb (kind stem);
6) secondary kind stem: when temperature gos up, be planted in by the one-level kind stem of acquisition in the matrix of vermiculite, humus soil and the mixing of thin bark, vermiculite, humus soil and thin bark volume ratio are 3:1:1, and thin bark particle diameter is less than 1cm, to drench 1000 times of carbendazols, conveniently manage; When kind of a stem sends sprouting, start every first quarter moon spray one time of nutrition liquid, bulb germination survival rate more than 90%, until blade is withered and yellow come off time, underground part bletilla striata bulb of gathering, obtains and produces with planting the secondary kind stem (kind stem) of stem as cultivation.
Claims (1)
1. a production method for bletilla seed stem, is characterized in that: comprise the steps:
1) process of explant and inoculation: gather bletilla striata capsule in the 9-10 month, surface dirt is rinsed well with water, naturally after drying, superclean bench is alcohol-pickled sterilization 30 s of 75% with mass percent concentration, use 0.1% mercuric chloride liquid soaking disinfection 8 min again, then after using sterile water wash 5 times, blot with the moisture of aseptic filter paper by bletilla striata capsule surface again, after seed in bletilla striata capsule is taken out, broadcast sowing the surface of seed germination medium uniformly, cultivate in culturing room, cultivation temperature is 25 ± 2 DEG C, intensity of illumination is 1500-2000Lx, light application time is 12h/d, the main body of described germination medium is 1/2MS medium, and wherein NAA is 0.5mg/L, 6-BA is 1.0 mg/L, and the mass percent of white sugar is 3%, and the mass percent of agar powder is 0.7%, pH is 5.8,
2) acquisition of protocorm and propagation: seed culture is after 10 days, form yellow protocorm, be transferred to when protocorm does not differentiate blade in proliferated culture medium, be positioned over culturing room's cultivation that temperature is 25 ± 2 DEG C, intensity of illumination 1500-2000Lx, light application time 12h/d, cultivated after 25 days, formed shape protocorm of growing thickly; The main body of described proliferated culture medium is MS medium, and wherein NAA is 0.2mg/L, 6-BA is 0.5 mg/L, and the mass percent of white sugar is 3%, and the mass percent of agar powder is 0.7%, pH is 5.8;
3) protocorm differentiation seedling: shape protocorm of growing thickly is transferred in differential medium, puts the culturing room of 25 ± 2 DEG C, intensity of illumination 1500-2000Lx, light application time 12h/d CMC model, to cultivating the plant obtaining 2-3 sheet leaf; The main body of described differential medium is MS medium, and wherein NAA is 0.3mg/L, and the mass percent of white sugar is 3%, and the mass percent of agar powder is 0.7%, pH is 5.8;
4) acquisition of cormel in vitro: the plant that step 3) obtains is transferred in root media, the culturing room being 25 ± 2 DEG C in temperature cultivates, and intensity of illumination 1500-2000Lx, light application time 12h/d, cultivated after 45-60 days, obtains cormel in vitro; The main body of described root media is MS medium, and wherein NAA is 0.5mg/L, 6-BA is 0.1mg/L, and the mass percent of white sugar is 3%, and the mass percent of agar powder is 0.7%, pH is 5.8;
5)
onelevel plants the acquisition of stem: the blake bottle being formed with cormel in vitro is placed on ambient temperatare and puts 3 days, then sealed membrane hardening is opened 2 days, with tweezers, the cormel in vitro in bottle is pressed from both sides out, medium is rinsed out under running water, and clean out dead leaf and root, plant in the mixed-matrix of ready vermiculite and thin bark, the volume ratio of vermiculite and thin bark is 4:1, thin bark particle diameter is less than 1cm, water 1000 times of carbendazols of drenching, then conveniently manage, start every first quarter moon during sprouting pending and spray one time of nutrition liquid; Treat that vegetative period terminates, blade is withered and yellow to come off, the bletilla striata bulb of underground part of gathering, and namely obtains one-level kind bulb;
6) secondary kind stem: when temperature gos up, be planted in by the one-level kind stem of acquisition in the matrix of vermiculite, humus soil and the mixing of thin bark, the volume ratio of vermiculite, humus soil and thin bark is 3:1:1, and thin bark particle diameter is less than 1cm, to drench 1000 times of carbendazols, conveniently manage; When kind of a stem sends sprouting, start every first quarter moon spray one time of nutrition liquid, until blade is withered and yellow come off time, underground part bletilla striata bulb of gathering, obtains and produces with planting the secondary kind stem of stem as cultivation.
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Families Citing this family (14)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103947425B (en) * | 2014-04-28 | 2015-08-05 | 黔西南州凌飞中草药开发有限公司 | A kind of cultivation implantation methods of bletilla |
| CN103947433B (en) * | 2014-05-12 | 2015-11-04 | 贵州省农作物品种资源研究所 | A kind of bletilla group training bulb forming seedling through one step culture method and matrix thereof |
| CN104054565B (en) * | 2014-06-13 | 2016-04-20 | 江苏茅山地道中药材种植有限公司 | A kind of hardening matrix improving bletilla striata plantlet in vitro survival rate |
| 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 |
| CN104255496B (en) * | 2014-09-23 | 2016-03-16 | 江苏农林职业技术学院 | A kind of method of Fast-propagation bletilla striata test-tube plantlet |
| CN104335903B (en) * | 2014-11-21 | 2017-04-05 | 广西中医药大学 | It is a kind of to promote Pseudobulbus Bletillae (Rhizoma Bletillae) rapid propagation method |
| CN104719152B (en) * | 2015-02-16 | 2017-04-19 | 贵州省农作物品种资源研究所 | Rhizoma bletillae industrialized seedling method |
| CN105145361B (en) * | 2015-09-17 | 2017-06-30 | 康美药业（文山）药材种植管理有限公司 | A kind of method of reproducing bletilla striata tissue-cultured seedling and the implantation methods of the bletilla striata |
| CN105028215B (en) * | 2015-09-17 | 2017-11-14 | 康美药业（文山）药材种植管理有限公司 | It is a kind of for the root media of bletilla striata tissue culture and the tissue culture method of the bletilla striata |
| CN105123529A (en) * | 2015-09-22 | 2015-12-09 | 宜昌市农业科学研究院 | Rapid propagation and efficient cultivation method of Bletilla striata |
| CN107371710B (en) * | 2017-08-08 | 2020-04-17 | 云南农业大学 | Bletilla striata seed direct seeding two-stage seedling method |
| CN109169274A (en) * | 2018-09-03 | 2019-01-11 | 云南中医学院 | A kind of method of rejuvenation in bletilla test tube seedling bottle |
| CN109717078A (en) * | 2019-03-05 | 2019-05-07 | 江西农业大学 | A kind of bletilla striata cormel in vitro growth cultural method |
| CN110447540A (en) * | 2019-09-11 | 2019-11-15 | 遵义医科大学 | A kind of tissue cultivating method of bletilla seedling |
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| CN102283115A (en) * | 2011-06-28 | 2011-12-21 | 陕西科技大学 | Method for quickly reproducing bletilla striata seedlings |
| CN103314855A (en) * | 2013-07-08 | 2013-09-25 | 重庆市秀山红星中药材开发有限公司 | Bletilla seed tissue culture propagation method |
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