CN105075861A - Bletilla striata embryogenic cytogenesis and cotyledon embryo culturing method - Google Patents
Bletilla striata embryogenic cytogenesis and cotyledon embryo culturing method Download PDFInfo
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- CN105075861A CN105075861A CN201510482911.7A CN201510482911A CN105075861A CN 105075861 A CN105075861 A CN 105075861A CN 201510482911 A CN201510482911 A CN 201510482911A CN 105075861 A CN105075861 A CN 105075861A
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Abstract
The invention discloses a bletilla striata embryonic cell obtaining and multiplying and cotyledon embryo culturing method. The method comprises the steps of obtaining of bletilla striata seed embryos, screening and multiplying of embryonic cells, liquid subculture of the embryonic cells and regeneration and growing of cotyledon embryos. According to the method, the bletilla striata seed embryos are induced to form embryonic callus, the embryonic cells are fast multiplied in a liquid culture mode, a specific solid medium is coated with the embryonic cells to regenerate bletilla striata cotyledon embryos, bletilla striata seedlings with genetic stability and uniform quality can be fast cultivated within a short time, and large-scale industrial artificial cultivation of bletilla striata can be achieved through transplanting. The problem that bletilla striata resources are in shortage can be effectively relieved, and wide market prospects are achieved.
Description
Technical field
The invention belongs to herbaceous plant artificial breeding and technical field of cultivation, particularly relate to a kind of bletilla striata embryo somatic cells and occur and cotyledonary embryos cultural method.
Background technology
The bletilla striata (
bletillastriata(Thunb.) Reichb.f.) also make bletilla, Lian Jicao, Gan Gen, for the orchid family bletilla striata belongs to herbaceos perennial, be distributed in China, Korea, Japan, the ground such as East China, south China, Henan, Shaanxi, Sichuan, Yunnan are originated in China.The stem tuber of the bletilla striata is China's traditional Chinese medicine, has the effects such as tonifying lung, detumescence, myogenic, hemostasis, sore, can be used for the diseases such as treatment lung hinders hemoptysis, metal-inflicted wound is hemorrhage, canker sores, soup fire are burnt, rhagadia manus et pedis.In addition, bletilla striata pattern is gorgeous, and be a kind of well landscape plant, therefore market is very vigorous to the demand of the bletilla striata.
Along with the sharply expansion of the market demand, the wild bletilla striata degree of meeting with of China's most area is excavated in recent years, causes its wild natural resources sharply to reduce, endangered, by one of country's Wild Medicinal being classified as focused protection.For protection bletilla striata resource, artificial breeding and the cultivation of the bletilla striata are imperative.But the seed due to the bletilla striata is very tiny and without endosperm, be therefore difficult to sprout and growth under natural situation, traditional cultivation is mainly by division propagation, but the division propagation cycle is long, and reproductive efficiency is low, and consumption kind of amount is large, is difficult to the needs meeting a large amount of cultivation.
1902, German botanist breathed out John Berendt prophesy: any one cell of plant corpus has the potential ability growing up to complete individuals, and this potential ability is just the totipotency of plant cell.1958, the Si Diwute of the U.S., at the root cells cultivating wild carrot, obtained the whole plant from individual cells finally.The seventies, American Japanese scholars Mu has loose bowels lattice through studying the entire work flow summing up factory's breeding plant, and after this industrialized propagation plant is widely used.As the famous flowers such as G. paniculata (babysbreath), tulip, carnation have bred by this method in Holland; China also establishes such flowers factory, and we also carry out testing and succeeding on the crops such as tobacco, rape, tomato.Plant cell and tissue culture technology has following characteristics: (1) condition of culture can artificial adjustment; (2) growth cycle is short, and reproduction rate is high; (3) convenient management, is beneficial to factorial praluction and Automated condtrol.
This technology is significant in the protection of endangered plants kind; tissue cultures mode at present about the bletilla striata has bletilla striata seeds axenic germination method; the method for tissue culture etc. of bletilla striata stem tuber, lateral bud, stem apex or young root; induced embryonic callus is carried out about utilizing bletilla striata seeds body early embryo; and carry out the breeding of embryo somatic cells by liquid rapid cellular proliferation mode, thus regeneration cotyledonary embryos finally form plantlet there is no report.
Summary of the invention
The object of this invention is to provide a kind of bletilla striata cellular liquid and cultivate the method forming cotyledonary embryos.The problems such as current bletilla striata wild resource is in imminent danger and Traditional Man cultivation method exists Genomic instability, the cycle is long, reproductive efficiency is low in the hope of effectively solving, consumption kind of amount is large.
Preparation process of the present invention is as follows:
(1) carry out specialty to bletilla striata prematurity capsule to disinfect, and aseptically, strip off bletilla striata capsule shell obtains bletilla striata seeds;
(2) under the bletilla striata seeds that step (1) obtains being placed in Stereo microscope, remove seed coat, be separated the body early embryo of bletilla striata seeds;
(3) the VW medium put into by the bletilla striata embryonic cell that step (2) obtains containing variable concentrations hormone carries out induction and the breeding of cells,primordial;
(4) cells,primordial that step (3) obtains is carried out the cultivation of liquid suspension passage with rapid, high volume propagation bletilla striata cells,primordial;
(5) cells,primordial that step (4) obtains is coated the VW solid culture medium being added with ABA and cultivate bletilla striata cotyledonary embryos.
Wherein:
Specialty described in step (1) disinfect into: first with water by clean for bletilla striata prematurity capsule surface clean, then with liquid detergent washing 5 ~ 10min, put running water 20 ~ 30min, use 75% alcohol surface sterilization 5 ~ 10min again, sterile water wash 4 ~ 6 times, use 0.1-0.2% mercuric chloride solution soaking disinfection 5 ~ 10min again, sterile water wash 5 ~ 7 times, then suck surface moisture with aseptic filter paper.
Fiber differentiation based formulas in step (3) is: VW medium, in addition containing 2,4-dichlorphenoxyacetic acid (2,4-D) 1 ~ 5mg/L, 6-benzylaminopurine (6-BA) 0.1 ~ 0.5mg/L, sucrose 4% ~ 6%, agar 5.2%.
Cells,primordial inductive condition in step (3) is: remain on 25 ± 2 DEG C in temperature, induction between completely unglazed light culture, switching is started after 25 ~ 35 days, continuous switching 3 times, distinguishes embryo callus and non embryogenic callus according to the external form of callus and callus gloss and screens embryo callus cell one by one.
The medium that in step (4), liquid suspension culture is used is: 2/3VW liquid nutrient medium, in addition containing zeatin (ZT) 0.4 ~ 0.6mg/L, α-naphthaleneacetic acid (NAA) 0.05 ~ 0.1mg/L; Condition of culture is: shaking table frequency of oscillation is 100 ~ 120rpm, temperature 25 DEG C, and lucifuge darkroom is cultivated.
The solid culture medium cultivating bletilla striata cotyledonary embryos in step (5) is: VW solid culture medium, in addition containing abscisic acid (ABA) 0.1 ~ 0.3mg/L; Condition of culture is: the indoor cultivation of the illumination cultivation that room temperature is 25 ± 2 DEG C 20 ~ 30 days.
Use the present invention, can be implemented in quickly breeding in the short time and go out inheritance stability and the homogeneous bletilla striata cotyledonary embryos seedling of quality, again by transplanting the artificial planting that can realize the heavy industrialization of the bletilla striata, effectively can alleviate the problem of bletilla striata resource scarcity, market prospects are boundless.
Embodiment
Below in conjunction with embodiment, the present invention is further elaborated, and those of ordinary skill in the art in scope of the present invention and essence, can carry out various accommodation and amendment to the present invention.
Embodiment 1:
Immature bletilla striata capsule is chosen in collection, with water by clean for its surface clean, 5min is washed with liquid detergent, put running water 20min, use 75% alcohol surface sterilization 5min again, sterile water wash 4 times, then use 0.1-0.2% mercuric chloride solution soaking disinfection 5min, sterile water wash 5 times, then sucks surface moisture with aseptic filter paper; Aseptically, strip the floury bletilla striata seeds in capsule, under bletilla striata seeds being placed in the stereomicroscope of superclean bench, under 400 times of mirrors, remove seed coat, be separated the embryo of bletilla striata seeds; Bletilla striata seeds embryo is put into containing 2,4-dichlorphenoxyacetic acid (2,4-D) 1mg/L, 6-benzylaminopurine (6-BA) 0.15mg/L, sucrose 4%, agar 5.2%, VW medium in carry out the induction of embryo callus, be positioned between light culture that environmental temperature remains the complete unglazed photograph of 25 ± 2 DEG C and cultivate 35 days, periodically switching bletilla striata callus, continuously switching 3 times; While switching, distinguish embryo callus and non embryogenic callus according to the external form of callus, color and callus gloss, filter out embryo callus; The embryo callus of screening is transferred in the 500mL conical flask of dress 100mL containing the 2/3VW liquid proliferated culture medium of zeatin (ZT) 0.6mg/L, α-naphthaleneacetic acid (NAA) 0.08mg/L and carry out periodic Secondary Culture, every conical flask inoculates the bletilla striata embryo callus of about 2g, postvaccinal conical flask is put in shaking table, shaking table frequency of oscillation is 100rpm, temperature 25 DEG C, after lucifuge darkroom cultivates 5 days, suspension DNA concentration increases to 4 times of original concentration; By the bletilla striata embryo somatic cells of liquid suspension culture through aseptic filtration, filtering liquid nutrient medium, aseptically embryo somatic cells is coated (concentration of ABA is 0.1mg/L) on the VW solid culture medium containing abscisic acid (ABA), be positioned over the indoor cultivation of illumination cultivation 20 days of room temperature 24 DEG C, the cotyledonary embryos of the visible bletilla striata occurs.
Embodiment 2:
Gather the bletilla striata capsule choosing mature and plump, with water by clean for its surface clean, 8min is washed with liquid detergent, put running water 25min, use 75% alcohol surface sterilization 8min again, sterile water wash 5 times, then use mercuric chloride solution soaking disinfection 8min, sterile water wash 6 times, then sucks surface moisture with aseptic filter paper; Aseptically, strip the floury bletilla striata seeds in capsule, under bletilla striata seeds being placed in the stereomicroscope of superclean bench, under 400 times of mirrors, remove seed coat, be separated the embryo of bletilla striata seeds; Bletilla striata seeds embryo is put into containing 2,4-dichlorphenoxyacetic acid (2,4-D) 2mg/L, 6-benzylaminopurine (6-BA) 0.3mg/L, sucrose 6%, agar 5.2%, VW medium in carry out the induction of embryo callus, be positioned between light culture that environmental temperature remains the complete unglazed photograph of 25 DEG C and cultivate 28 days, periodically switching bletilla striata callus, continuously switching 3 times; While switching, distinguish embryo callus and non embryogenic callus according to the external form of callus and callus gloss, filter out embryo callus; The embryo callus of screening is transferred in the 500mL conical flask of dress 100mL containing the 2/3VW liquid proliferated culture medium of zeatin (ZT) 0.5mg/L, α-naphthaleneacetic acid (NAA) 0.06mg/L and carry out periodic Secondary Culture, every conical flask inoculates the bletilla striata embryo callus of about 20-50g, postvaccinal conical flask is put in shaking table, shaking table frequency of oscillation is 110rpm, temperature 25 DEG C, after lucifuge darkroom cultivates 7 days, suspension DNA concentration increases to 4 times of original concentration; By the bletilla striata embryo somatic cells of liquid suspension culture through aseptic filtration, filtering liquid nutrient medium, aseptically embryo somatic cells is coated (concentration of ABA is 0.2mg/L) on the VW solid culture medium containing abscisic acid (ABA), be positioned over the indoor cultivation of illumination cultivation 25 days of room temperature 25 DEG C, the cotyledonary embryos of the visible bletilla striata occurs.
Embodiment 3:
Gather the bletilla striata capsule choosing mature and plump, with water by clean for its surface clean, 10min is washed with liquid detergent, put running water 30min, use 75% alcohol surface sterilization 10min again, sterile water wash 6 times, then use mercuric chloride solution soaking disinfection 10min, sterile water wash 7 times, then sucks surface moisture with aseptic filter paper; Aseptically, strip the floury bletilla striata seeds in outstanding achievement, under bletilla striata seeds being placed in the stereomicroscope of superclean bench, under 400 times of mirrors, remove seed coat, be separated the embryo of bletilla striata seeds; Bletilla striata seeds embryo is put into containing 2,4-dichlorphenoxyacetic acid (2,4-D) 5mg/L, 6-benzylaminopurine (6-BA) 0.3mg/L, sucrose 6%, agar 5.2%, VW medium in carry out the induction of embryo callus, be positioned between light culture that environmental temperature remains the complete unglazed photograph of 26 DEG C and cultivate 25 days, periodically switching bletilla striata callus, continuously switching 3 times; While switching, distinguish embryo callus and non embryogenic callus according to the external form of callus and callus gloss, filter out embryo callus; The embryo callus of screening is transferred in the 500mL conical flask of dress 100mL containing the 2/3VW liquid proliferated culture medium of zeatin (ZT) 0.4mg/L, α-naphthaleneacetic acid (NAA) 0.1mg/L and carry out periodic Secondary Culture, every conical flask inoculates the bletilla striata embryo callus of about 50g, postvaccinal conical flask is put in shaking table, shaking table frequency of oscillation is 120rpm, temperature 25 DEG C, after lucifuge darkroom cultivates 8 days, suspension DNA concentration increases to 4 times of original concentration; By the bletilla striata embryo somatic cells of liquid suspension culture through aseptic filtration, filtering liquid nutrient medium, aseptically embryo somatic cells is coated (concentration of ABA is 0.3mg/L) on the VW solid culture medium containing abscisic acid (ABA), be positioned over the indoor cultivation of illumination cultivation 30 days of room temperature 26 DEG C, the cotyledonary embryos of the visible bletilla striata occurs.
For the VW basal medium formulation of Plant Tissue Breeding in table 1.
Table 1
Claims (6)
1. bletilla striata cells,primordial occurs and a cotyledonary embryos cultural method, it is characterized in that comprising the steps:
(1) carry out specialty to bletilla striata prematurity capsule to disinfect, and aseptically, strip off bletilla striata capsule shell obtains bletilla striata seeds;
(2) under the bletilla striata seeds that step (1) obtains being placed in Stereo microscope, remove seed coat, be separated the body early embryo of bletilla striata seeds;
(3) the VW medium put into by the bletilla striata embryonic cell that step (2) obtains containing variable concentrations hormone carries out induction and the breeding of cells,primordial;
(4) cells,primordial that step (3) obtains is carried out the cultivation of liquid suspension passage with rapid, high volume propagation bletilla striata cells,primordial;
(5) the embryo somatic cells that step (4) obtains is coated the VW solid culture medium being added with ABA and cultivate bletilla striata cotyledonary embryos.
2. bletilla striata cells,primordial according to claim 1 occurs and cotyledonary embryos cultural method, it is characterized in that: the specialty described in step (1) disinfect into: first with water by clean for bletilla striata capsule surface clean, then with liquid detergent washing 5 ~ 10min, put running water 20 ~ 30min, use 75% alcohol surface sterilization 5 ~ 10min again, sterile water wash 4 ~ 6 times, then use mercuric chloride solution soaking disinfection 5 ~ 10min, sterile water wash 5 ~ 7 times, then sucks surface moisture with aseptic filter paper.
3. bletilla striata cells,primordial according to claim 1 occurs and cotyledonary embryos cultural method, it is characterized in that: the Fiber differentiation based formulas in step (3) is: VW medium, in addition containing 2,4-dichlorphenoxyacetic acid (2,4-D) 1 ~ 5mg/L, 6-benzylaminopurine (6-BA) 0.1 ~ 0.5mg/L, sucrose 4% ~ 6%, agar 5.2%.
4. bletilla striata cells,primordial according to claim 1 occurs and cotyledonary embryos cultural method, it is characterized in that: the cells,primordial inductive condition in step (3) is: remain on 25 ± 2 DEG C in temperature, induction between completely unglazed light culture, switching is started after 25 ~ 35 days, continuous switching 3 times, distinguishes embryo callus and non embryogenic callus according to the external form of callus, color and callus gloss and screens embryo callus cell one by one.
5. bletilla striata cells,primordial according to claim 1 occurs and cotyledonary embryos cultural method, it is characterized in that: the middle liquid ` of step (4) cultivates medium used and is: 2/3VW liquid nutrient medium, in addition containing zeatin (ZT) 0.4 ~ 0.6mg/L, α-naphthaleneacetic acid (NAA) 0.05 ~ 0.1mg/L; Condition of culture is: shaking table frequency of oscillation is 100 ~ 120rpm, temperature 25 DEG C, and lucifuge darkroom is cultivated.
6. bletilla striata cells,primordial according to claim 1 occurs and cotyledonary embryos cultural method, it is characterized in that: the solid culture medium cultivating bletilla striata cotyledonary embryos in step (5) is: VW solid culture medium, in addition containing abscisic acid (ABA) 0.1 ~ 0.3mg/L; Condition of culture is: the indoor cultivation of the illumination cultivation that room temperature is 25 ± 2 DEG C 20 ~ 30 days.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105830582A (en) * | 2016-04-15 | 2016-08-10 | 成都大学 | Quickbackfillcultivation method of rhizoma bletillae seeds |
| CN109917034A (en) * | 2019-03-19 | 2019-06-21 | 遵义医科大学 | The quantitative detecting method of 2,7- dihydroxy -4- methoxyl group -9,10- dihydro phenanthrene in a kind of bletilla suspended culture cell |
-
2015
- 2015-08-10 CN CN201510482911.7A patent/CN105075861A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105830582A (en) * | 2016-04-15 | 2016-08-10 | 成都大学 | Quickbackfillcultivation method of rhizoma bletillae seeds |
| CN105830582B (en) * | 2016-04-15 | 2018-05-01 | 成都大学 | A kind of the quick of bletilla seed returns native breeding method |
| CN109917034A (en) * | 2019-03-19 | 2019-06-21 | 遵义医科大学 | The quantitative detecting method of 2,7- dihydroxy -4- methoxyl group -9,10- dihydro phenanthrene in a kind of bletilla suspended culture cell |
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