CN104920208B - A kind of bletilla striata tissue-culturing rapid propagation culture medium - Google Patents
A kind of bletilla striata tissue-culturing rapid propagation culture medium Download PDFInfo
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- CN104920208B CN104920208B CN201510253683.6A CN201510253683A CN104920208B CN 104920208 B CN104920208 B CN 104920208B CN 201510253683 A CN201510253683 A CN 201510253683A CN 104920208 B CN104920208 B CN 104920208B
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Abstract
The invention discloses a kind of bletilla striata tissue-culturing rapid propagation culture medium, including bletilla striata seeds sowing culture medium, subculture medium and field planting culture medium of taking root.Compared with prior art, the present invention has the advantages that germination rate is high, incubation time is short, the tissue-cultured seedling bulb of acquisition is big, rooting efficiency is good, transplanting survival rate is high, with good market prospects.
Description
Technical field
The present invention relates to the field of tissue culture of plant, and in particular to a kind of culture medium prescription of bletilla striata tissue-culturing rapid propagation.
Background technology
The bletilla striata,（Scientific name：Bletilla striata）Also known as Lian Jicao, Gan Gen, give free of charge, indocalamus orchid, Zhu Lan, purple blue, purple a species of orchid,
Hundred large bamboo hats with a conical crown and broad brim, are that the orchid family bletilla striata belongs to herbaceos perennial, are distributed widely in Yangtze river basin each province of China, main product is in Guizhou, Sichuan, lake
The ground such as south, Hubei, Anhui, Henan, Zhejiang, Shaanxi.The bletilla striata not only has higher ornamental value, is also the important Chinese medicine of China
One of material, is used as medicine with stem tuber, energy astringing to arrest bleeding, detumescence and promoting granulation, is widely used in treatment pulmonary tuberculosis and bronchiectasis hemoptysis, stomach
The diseases such as ulcer bleeding.
For a long time, bletilla striata market relies primarily on wild resource, with demand increase and price it is surging, cause
Wild resource is increasingly exhausted.Therefore, it is the key for alleviating the demand and supply contraction to solve bletilla striata seedling quick breeding technology.It is existing
Solid medium is seeded into using seed more than bletilla striata seedling tissue-culturing rapid propagation, then goes out tissue-cultured seedling through final production of breaking up and take root.
However, existing bletilla striata group culturation rapid propagating technology need further in terms of seed germination rate, tissue culture seedling rooting and bulb size
Improve.
The content of the invention
The technical problems to be solved by the invention are：There is provided a kind of training of bletilla striata tissue-culturing rapid propagation in view of the shortcomings of the prior art
Base is supported, to improve seed germination rate, the tissue-cultured seedling bulb size for shortening incubation time, improving acquisition, rooting rate be improved, so as to fast
Fast reproducing bletilla striata tissue-cultured seedling, meets market needs.Therefore, the present invention uses following technical scheme：
A kind of bletilla striata tissue-culturing rapid propagation culture medium, it is characterised in that it include bletilla striata seeds sowing culture medium, subculture medium,
Take root field planting culture medium；
Bletilla striata seeds sow culture medium using MS culture mediums as minimal medium, contain in every liter of matrix：Agar 8g, white sugar
30g, coconut milk 80ml, activated carbon 0.5g, 6-BA0.05mg, NAA 0.25mg, the pH value of culture medium is 5.4；
The subculture medium contains using MS culture mediums as minimal medium in every liter of matrix：Agar 8g, white sugar 30g,
Coconut milk 80ml, activated carbon 0.5g, 6-BA 0.5mg, NAA 0.1mg, the pH value of culture medium is 5.4；
The field planting culture medium of taking root contains in every liter of matrix：Spend precious No. I 2 g, spend precious II 1g, agar 8g, white sugar
20g, activated carbon 1g, banana meat 40g, mashed potato 20g, the pH value of culture medium is 5.4.
Hua BaoNumber and spend treasuredNumber precious Products are spent for the U.S.（It is limited that Taiwan produced in USA closes gardening enterprise stock
Company dispenses, HYPONEX）, it is the water-soluble fertilizer of quick-acting pulvis；Hua BaoNumber composition be N：P：K, quality proportioning is 7：6：19, flower
It is preciousNumber composition be N：P：K, quality proportioning is 20：20：20.N, K, P refer respectively to NO3Nitrate nitrogen, PO3Phosphorous oxide, KO3
Potassium oxide.
The preparation method of the coconut milk is that fresh coconut palm fruit takes its juice.
The preparation method of the banana meat is its pulp of banana peeling and taking.
The preparation method of the potato is that potato is cleaned peeling, in mass ratio 1:5 ratio adds water, and is taken after boiling
Go out potato ball and stir into pasty state with mixer to form.
NAA is the abbreviation of methyl α-naphthyl acetate (1-Naphthaleneacetic acid), is a kind of nothing for being soluble in organic solvent
Color solid；It is a kind of plant hormone auxin, is usually used in the root of hair powder of commercialization or rooting agent.
6-BA is the abbreviation of 6- benzyl aminoadenines, and it is a kind of basic element of cell division, and main function is the formation for promoting bud,
Can also evoked callus generation.
Bletilla striata seeds sowing culture medium, subculture medium, the field planting culture medium of taking root of the present invention is used based on MS
Culture medium, MS（MURASHIGE & SKOOG）Culture medium is to use most common culture medium at present, and it has higher inorganic
Salinity, the mineral nutrition needed for ensure that tissue growth can also accelerate the growth of callus.
The present invention is relative to prior art, with advantages below：
（1）Bletilla striata seeds nature germination rate is extremely low, using this sowing culture medium its germination rate can be made to reach more than 95%,
Substantially increase the seed germination rate of the bletilla striata.
（2）Sowed in the present invention in culture medium and subculture medium and with the addition of coconut milk, coconut milk contains several amino acids
And vitamin etc., the trace element and organic substance of abundance are provided for bletilla tissue culture seedlings, the bletilla striata seedling of acquisition is in plant height and leaf
Existing culture medium is superior in terms of piece size.
（3）Field planting culture medium of being taken root in the present invention, which is used, to be spent precious No. I and spends precious No. II water-soluble fertilizer, what the recipe ratio was commonly used
MS culture mediums match somebody with somebody convenient, and with the addition of the banana meat of hestening rooting and promote the potato of strong sprout, the bletilla striata seedling of acquisition
Take root, bulb is big, robust plant, transplanting survival rate is up to more than 95%.
（4）The present invention is sowed by bletilla striata seeds, squamous subculture and culture of rootage can obtain a large amount of bletilla striata groups in a short time
Seedling is trained, after seed sprouts culture 40 days, subculture medium is transferred to, root media is transferred to after 50 days, can be by acquisition after 60 days
Rooted seedling carries out hardening.
Brief description of the drawings
Fig. 1 is the change schematic diagram in kind that the bletilla striata is cultivated using fast breeding culture medium of the present invention.
Embodiment
Embodiment 1
1）With culture medium：
Every liter of sowing culture medium is composed of the following components：Agar 8g+ white sugar 30g+ coconut waters 80ml+6-BA0.05mg+NAA
0.25mg, remaining is MS, pH=5.4.
Every liter of subculture medium is composed of the following components：Agar 8g+ white sugar 30g+ coconut water 80ml+ activated carbons 0.5g+6-
BA 0.5mg+NAA 0.1mg, remaining is MS, pH=5.4.
Every liter of field planting culture medium of taking root is composed of the following components：Spend precious I 2g+spend precious II 1g+ agar 8g+ white sugar 20g
+ activated carbon 1g+ banana meat 40g+ mashed potato 20g, pH=5.4.
2）Explant is selected：Choose healthy and strong wild bletilla striata capsule.
3）Sterilizing：（1）Capsule rinses 30min with flowing water；（2）With aseptic water washing 1 time；（3）75% alcohol disinfecting 30s；
（4）0.1% mercuric chloride solution soaks 10min；（5）Aseptic water washing 3-5 times.
4）Inoculation：The capsule sterilized is placed on aseptic filter paper after suck dry moisture from middle part of fruit and peeled off, seed is fast
Speed is inoculated on sowing culture medium.
5）Seed sprouts culture：By the blake bottle being inoculated with（See Figure 1A）Culturing room's culture is transferred to, condition of culture is：Temperature
23-25 DEG C, illumination：1500-2000lx, lighting delay number：12h/d.
6）Squamous subculture：After seed sprouts culture 20 days, embryo is expanded substantially, and green is changed into from yellow, is broken up after 40 days
For bletilla striata seedling（See Figure 1B）.Bletilla striata seedling is transferred on subculture medium and cultivated, condition of culture is：23-25 DEG C of temperature, light
According to：1500-2000lx, lighting delay number：12h/d.After squamous subculture 50 days, bletilla striata seedling can develop into the high seedlings of 1.5cm（See
Fig. 1 C）, every 50 days subcultures 1 time, obtain a large amount of bletilla striata seedlings.
7）Field planting of taking root is cultivated：3cm or so bletilla striata seedling is transferred on field planting culture medium of taking root and carries out culture of rootage, trained
Foster condition is：23-25 DEG C of temperature, illumination：1500-2000lx, lighting delay number：12h/d.After culture of rootage 60 days, bletilla striata seedling
It can grow to 8cm or so height, the long 5cm of root or so, 3-4 bars of averagely taking root, bulb diameter 0.5cm or so.（Fig. 1 D, E）
8）Transplant：The healthy tissue-cultured seedling for selecting Root Distribution uniform, sturdy, opens blake bottle hardening one week, afterwards by root
The agar medium in portion is rinsed well, is then transplanted to peat：Loose squama：Perlite=2:1:In 1 matrix.
Embodiment 2
Control group 1, bletilla striata tissue-culturing rapid propagation culture medium according to the literature, sowing culture medium uses the culture of document report
Based formulas, subculture medium and root media use this patent culture medium prescription, and culture medium prescription is as follows：
Every liter of sowing culture medium is composed of the following components：Agar 6.5g+ sucrose 30g+6-BA 1.0mg, remaining is 1/2MS,
pH=5.8。
Every liter of subculture medium is composed of the following components：Agar 8g+ white sugar 30g+ coconut water 80ml+ activated carbons 0.5g+6-
BA 0.5mg+NAA 0.1mg, remaining is MS, pH=5.4.
Every liter of field planting culture medium of taking root is composed of the following components：Spend precious I 2g+spend precious II 1g+ agar 8g+ white sugar 20g
+ activated carbon 1g+ banana meat 40g+ mashed potato 20g, pH=5.4.
The operation be the same as Example 1 such as explant selection, sterilizing, inoculation, transplanting.
Embodiment 3
Control group 2, bletilla striata tissue-culturing rapid propagation culture medium according to the literature, sowing culture medium and root media are using this
Patent media is formulated, and subculture medium uses the culture medium prescription of document report, and culture medium prescription is as follows：
Every liter of sowing culture medium is composed of the following components：Agar 8g+ white sugar 30g+ coconut waters 80ml+6-BA0.05mg+NAA
0.25mg, remaining is MS, pH=5.4.
Every liter of subculture medium is composed of the following components：Agar 6.5g+ sucrose 30g+6-BA 1.0mg+NAA 0.15mg,
Remaining is MS, pH=5.8.
Every liter of field planting culture medium of taking root is composed of the following components：Spend precious I 2g+spend precious II 1g+ agar 8g+ white sugar 20g
+ activated carbon 1g+ banana meat 40g+ mashed potato 20g, pH=5.4.
The operation be the same as Example 1 such as explant selection, sterilizing, inoculation, transplanting.
Embodiment 4
Control group 3, bletilla striata tissue-culturing rapid propagation culture medium according to the literature, sowing culture medium and subculture medium are using this
Patent media is formulated, and root media uses the culture medium prescription of document report, and culture medium prescription is as follows：
Every liter of sowing culture medium is composed of the following components：Agar 8g+ white sugar 30g+ coconut waters 80ml+6-BA0.05mg+NAA
0.25mg, remaining is MS, pH=5.4.
Every liter of subculture medium is composed of the following components：Agar 8g+ white sugar 30g+ coconut water 80ml+ activated carbons 0.5g+6-
BA 0.5mg+NAA 0.1mg, remaining is MS, pH=5.4.
Every liter of field planting culture medium of taking root is composed of the following components：Agar 6.5g+ sucrose 30g+NAA 0.5mg+banana meat
75g, remaining is 1/2MS, pH=5.8.
The operation be the same as Example 1 such as explant selection, sterilizing, inoculation, transplanting.
Result of implementation：
Pass through the as shown by data of above example（Table 1）：All cultures of the embodiment 1 of application culture medium prescription of the present invention
Time substantially shortens than comparative examples 2,3,4；It is higher very than compareing using the sowing culture medium seed germination rate of patent of the present invention
It is many；The tissue-cultured seedling obtained in follow-up cultivation is in rooting rate, number of taking root, bulb size, leaf length, leaf width Ji Chu ping comprehensive states will
It is good a lot.
The not be the same as Example culture bletilla tissue culture seedlings situation table of table 1
| Incubation time | Seed germination rate | Rooting rate | Take root and count (bar) | Bulb diameter（cm） | Leaf is long（cm） | Leaf width（cm） | Miao Chu ping states | |
| Example 1 | 4 months | 96.5% | 97.6% | 4.8 | 0.5 | 2.1 | 0.5 | Growing way is uniform, and rhizome is sturdy |
| Example 2 | 5 months | 85.7% | 95.8% | 4.6 | 0.4 | 1.9 | 0.4 | Growing way is uniform |
| Example 3 | 6 months | 95.6% | 96.4% | 4.4 | 0.3 | 1.5 | 0.3 | Rhizome is not sturdy enough |
| Example 4 | 6 months | 95.4% | 85.5% | 3.4 | 0.2 | 1.5 | 0.3 | Rhizome is not sturdy enough |
Transplanting survival rate is counted after transplanting 2 months.All tissue culture transplantation of seedlings of embodiment 1 of application culture medium prescription of the present invention
Survival rate is compareed higher than embodiment 2,3,4, and transplanted seedling normal growth after 1 month, is physically well developed, robust growth.
The bletilla striata transplanted seedling growing state table that be the same as Example is not obtained of table 2
| Transplanting survival rate | Seedling growing way situation | Resistance | |
| Example 1 | 97.2% | Restoration ecosystem after transplanting 1 month, physically well develops | Pest and disease damage is few, robust growth |
| Example 2 | 95.8% | Restoration ecosystem after transplanting 1 month, physically well develops | Pest and disease damage is few, robust growth |
| Example 3 | 92.2% | Restoration ecosystem after transplanting 1.5 months | Pest and disease damage is less, and growing way is slightly weak |
| Example 4 | 85.3% | Restoration ecosystem after transplanting 1.5 months | Pest and disease damage is less, and growing way is slightly weak |
Therefore, bletilla striata seeds sowing culture medium, subculture medium, field planting culture medium three's collaboration of taking root in patent of the present invention
Preferably, some culture medium does not all reach optimal effectiveness to application effect during individually replacing bletilla striata tissue-culturing rapid propagation, it is proposed that by three
Culture medium is planted to develop and apply as an entirety.
Claims (1)
1. a kind of bletilla striata tissue-culturing rapid propagation culture medium, it is characterised in that it sows culture medium, subculture medium by bletilla striata seeds, taken root
It is colonized culture medium composition；
Bletilla striata seeds sow culture medium using MS culture mediums as minimal medium, contain in every liter of matrix：Agar 8g, white sugar 30g,
Coconut milk 80ml, activated carbon 0.5g, 6-BA0.05mg, NAA 0.25mg, the pH value of culture medium is 5.4；
The subculture medium contains using MS culture mediums as minimal medium in every liter of matrix：Agar 8g, white sugar 30g, coconut
Juice 80ml, activated carbon 0.5g, 6-BA 0.5mg, NAA 0.1mg, the pH value of culture medium is 5.4；
The field planting culture medium of taking root contains in every liter of matrix：Spend precious I 2g, spend precious II 1g, agar 8g, white sugar 20g, work
Property charcoal 1g, banana meat 40g, mashed potato 20g, the pH value of culture medium is 5.4.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105613286A (en) * | 2015-12-13 | 2016-06-01 | 武汉生物工程学院 | Method for rapidly producing bletilla striata test-tube plantlets |
| CN105766645B (en) * | 2016-03-31 | 2018-07-24 | 广西医科大学 | Bletilla tissue-cultured seedling efficient propagation method |
| CN105660422B (en) * | 2016-04-20 | 2017-09-29 | 遵义医学院 | A kind of method for tissue culture of bletilla seedling |
| CN105941160B (en) * | 2016-06-17 | 2018-06-12 | 四川农业大学 | A kind of breeding method of the bletilla striata |
| CN106417011A (en) * | 2016-08-30 | 2017-02-22 | 云南金碧制药有限公司 | Wild bletilla striata tissue culture rapid propagation method |
| CN108142296A (en) * | 2018-01-22 | 2018-06-12 | 黔东南民族职业技术学院 | A kind of efficient culture medium for sprouting bletilla striata seeds and its application |
| CN108207632B (en) * | 2018-01-22 | 2019-11-05 | 黔东南民族职业技术学院 | A kind of method of quick reproducing bletilla striata |
| CN108308029A (en) * | 2018-03-28 | 2018-07-24 | 普安县龙吟兴民种养殖开发有限公司 | A kind of bletilla striata tissue culture special culture media |
| 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 |
| CN109169275A (en) * | 2018-09-13 | 2019-01-11 | 甘肃源宜生物科技有限公司 | A kind of pale reddish brown trident bletilla striata tissue-culturing rapid propagation culture medium and method |
| CN111903530A (en) * | 2020-09-23 | 2020-11-10 | 宣威市福康生物科技有限公司 | Tissue culture seedling method for bletilla striata |
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| CN102972290B (en) * | 2012-11-15 | 2013-08-21 | 贵州师范大学 | Method for improving transplanting survival rate of rhizoma bletillae tissue culture seedling |
| 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 |
| CN103828715B (en) * | 2013-11-29 | 2017-01-18 | 安徽中升生物科技有限公司 | Bletilla striata tissue culture breeding method |
| 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 |
| CN104604680B (en) * | 2015-01-19 | 2016-08-17 | 杭州雷威农业开发有限公司 | Can promote that bletilla striata seeds sprouts the culture medium with growth of seedling and formula thereof and preparation method |
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Address after: Jiaxing City, Zhejiang province Haining city Changan town 314408 Chu village Jinzhu Park No. 1 Co-patentee after: KUNSHAN HONGYUE FLOWER CO., LTD. Patentee after: Yue Yue flower Limited by Share Ltd Address before: Jiaxing City, Zhejiang province Haining city Changan town 314408 Chu village Jinzhu Park No. 1 Co-patentee before: KUNSHAN HONGYUE FLOWER CO., LTD. Patentee before: Zhejiang Hongyue Horticultural Corporation |
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