CN106879466A - A kind of rapid propagation method of the Miaos medicinal materials green grass or young crops pod leaf - Google Patents
A kind of rapid propagation method of the Miaos medicinal materials green grass or young crops pod leaf Download PDFInfo
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- CN106879466A CN106879466A CN201710147110.4A CN201710147110A CN106879466A CN 106879466 A CN106879466 A CN 106879466A CN 201710147110 A CN201710147110 A CN 201710147110A CN 106879466 A CN106879466 A CN 106879466A
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- adventitious bud
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
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Abstract
The present invention relates to the method for Plant Tissue Breeding in agricultural biotechnologies, specifically, it is related to a kind of rapid propagation method of the Miaos medicinal materials green grass or young crops pod leaf, including the following steps for carrying out successively：Obtain explant, adventitious bud inducing, the squamous subculture of adventitious bud, the culture of rootage of adventitious bud, the test tube transplantation of seedlings of blue or green pod leaf；The inducing culture of described adventitious bud inducing includes：MS culture mediums, 0.5~1.0mg/L ZT, 0.1~0.3mg/L NAA, 25~30g/L sucrose, 3.5~4.0g/L agar, 0.5~1.0g/L activated carbons, pH is 5.4~5.8, and the Miaos medicinal materials green grass or young crops pod leaf test tube seedling transplanting survival rate of the method for the present invention reaches more than 91%.
Description
Technical field
The present invention relates to the method for Plant Tissue Breeding in agricultural biotechnologies, specifically, it is related to the Miaos medicinal materials green grass or young crops pod
The rapid propagation method of leaf.
Background technology
The Miaos medicinal materials rifle wall leaf is blue or green pod leaf (Helwingia japonica.japonica), is Cornaceae green grass or young crops pod leaf
Category machaka.Genus helwingia has blue or green pod leaf, Helwingia chinensis, the blue or green pod leaf in Tibet, Zhejiang green grass or young crops pod leaf and high eyebrow green grass or young crops pod leaf etc. 5
Kind, it is distributed mainly on Nepal, Sillim, Bhutan, north India, Upper Myanmar, North Vietnam, China and the Japan of east Asia
Deng state.5 kinds of China produces, and is distributed mainly on the original of more than 1500 meters of height above sea level such as Shaanxi, Henan, the Yangtze river basin and South China
In forest.Because blue or green pod leaf female flower grows without stalk, flower and fruit on the master pulse in blade face center, it is otherwise known as on Helwingia himalaica or leaf
Really.Its root, stem and leaf is used as medicine, with stimulating the circulation of the blood and cause the muscles and joints to relax, menstruation regulating, cough-relieving the effects such as, Miao ethnic group is among the people often with Helwingia himalaica, fig, China rose
Collocation treatment irregular menstruation, with Helwingia himalaica, short top top, Sedum uizoon collocation treatment internal lesion caused by overexertion, is used alone blue or green pod leaf older to treat
Cough and asthma.Additionally, blue or green pod leaf is machaka, dioecism, the flower of female plant and the fruit of staminiferous plant are respectively provided with very strong intuitive, this
Planting peculiar phenomenon can attract stopping for passerby to view and admire.On Landscape arrangement, be planted in dark and damp fertile place, such as plant lilac,
Under hall crabapple flower tree, or plant in before pool, wall side or step or potted plant in interior, there is good appreciation effect.
At present, blue or green pod leaf is cultivated in Guizhou, Guangxi Miao area on a small quantity still in introducing a fine variety the stage.Blue or green pod leaf is mainly used
Seminal propagation, but because its seed is often searched for food by birds, it is difficult to obtain the seed for being sufficiently used for breeding.Therefore, in order to solve green grass or young crops
The problem that its seedling lacks in pod leaf large-scale planting, it is necessary to set up the tissue culture quick breeding system of blue or green pod leaf, present invention choosing
Blue or green pod leaf stem-segment with node is selected for explant, it is real through adventitious bud induction culture, squamous subculture, culture of rootage and transplanting and other steps
The quick breeding of the Miaos medicinal materials green grass or young crops pod leaf is showed.The features such as present invention tool low cost, process is simple, fast seedling, can be directly used for
The factorial praluction of blue or green pod leaf seedling, genetic improvement and plasm resource protection for blue or green pod leaf have important practical significance.
At present, the report of Qing Jia leaf textures culture technique is at home and abroad there is no, does not also there is blue or green pod leaf tissue-culturing rapid propagation patent
Application.
The content of the invention
It is an object of the invention to provide a kind of rapid propagation method of the Miaos medicinal materials green grass or young crops pod leaf, the blue or green pod leaf band of present invention selection
Stem segment is explant, through adventitious bud induction culture, squamous subculture, culture of rootage and transplanting and other steps, realizes Miao ethnic group's medicine
The quick breeding of material green grass or young crops pod leaf, it is achieved thereby that the purpose of the present invention.
The present invention provide technical scheme be：A kind of rapid propagation method of the Miaos medicinal materials green grass or young crops pod leaf, including carry out successively
Following steps：Obtain explant, adventitious bud inducing, the squamous subculture of adventitious bud, the culture of rootage of adventitious bud, the examination of blue or green pod leaf
Pipe transplantation of seedlings；
The inducing culture of described adventitious bud inducing includes：MS culture mediums, 0.5~1.0mg/L ZT, 0.1~0.3mg/
L NAA, 25~30g/L sucrose, 3.5~4.0g/L agar, 0.5~1.0g/L activated carbons, pH are 5.4~5.8.
In the rapid propagation method of above-mentioned the Miaos medicinal materials green grass or young crops pod leaf, the method for described adventitious bud inducing is：By green grass or young crops
The explant of pod leaf is cleaned disinfect after be cut into the stem-segment with node of 1.0~1.5cm or so and be inoculated into inducing culture,
Induced synthesis adventitious bud by carrying out full light culture 25~30 days at 25~28 DEG C.
In the rapid propagation method of above-mentioned the Miaos medicinal materials green grass or young crops pod leaf, the squamous subculture used by the squamous subculture of adventitious bud
Base includes：MS culture mediums, 0.5~1.0mg/L 6-BA, 0.1~0.5mg/L NAA, 20~30g/L sucrose, 3.5~4.0g/L
Agar, 0.1~0.5g/L activated carbons, pH are 5.4~5.8.
In the rapid propagation method of above-mentioned the Miaos medicinal materials green grass or young crops pod leaf, the method tool of the squamous subculture of described adventitious bud
Body is：The adventitious bud obtained by adventitious bud inducing is cut into be about the stem section of 1~2cm and be inoculated into subculture medium and is cultivated
The subculture for being capable of achieving adventitious bud in 25~30 days.
In the rapid propagation method of above-mentioned the Miaos medicinal materials green grass or young crops pod leaf, the culture of rootage used by the culture of rootage of adventitious bud
Base includes：1/2MS, 0.1~0.3mg/L 6-BA, 0.1~0.3mg/L IBA, 0.5~1.0mg/L NAA, 15~20g/L sugarcanes
Sugar, 3.0~4.0g/L agar, pH are 5.4~5.8.
In the rapid propagation method of above-mentioned the Miaos medicinal materials green grass or young crops pod leaf, the method for the culture of rootage of described adventitious bud
For：The adventitious bud for being about 2.0~3.0cm that will be obtained by the squamous subculture of adventitious bud cuts from base portion and is inoculated into training of taking root
Culture can realize taking root for test tube seedling in 25~30 days in supporting base.
In the rapid propagation method of above-mentioned the Miaos medicinal materials green grass or young crops pod leaf, the method for described test tube transplantation of seedlings is specially：
The well-grown test tube seedling that will be obtained by the culture of rootage of adventitious bud cleans root in the natural light lower refining seedling 1~3 day in greenhouse
It is 1 that the culture medium in portion is transplanted in volume ratio:Seedling is cultivated in 1 fertile soil, bark mixed-matrix and obtains final product seedling.
In the rapid propagation method of above-mentioned the Miaos medicinal materials green grass or young crops pod leaf, described adventitious bud inducing, the subculture of adventitious bud
The condition of culture of culture is：Cultivation temperature is 25~28 DEG C, and intensity of illumination is 2000~2500lx, and light application time is 10~12
Hour/day.
In the rapid propagation method of above-mentioned the Miaos medicinal materials green grass or young crops pod leaf, the specific method of the explant of blue or green pod leaf is obtained
For：Robust growth is chosen in the wild, the stem-segment with node of disease-free blue or green pod leaf plant is explant, carries out water conservation after collection immediately
Moisturizing is processed.
Beneficial effect：
The present invention realizes the quick breeding of the Miaos medicinal materials green grass or young crops pod leaf, present invention tool cost using plant tissue culture technique
The features such as low, process is simple, fast seedling, survival rate to more than 91% can be directly used for the factorial praluction of blue or green pod leaf seedling.
Specific embodiment
With reference to specific embodiment, technical scheme is described in further detail, but do not constitute it is right
Any limitation of the invention.
Embodiment one
(1) explant collection：Robust growth is chosen in the wild, the stem-segment with node of disease-free blue or green pod leaf plant is explant
Body, carries out water conservation moisturizing treatment and takes back laboratory in time immediately after collection.
(2) adventitious bud inducing：First cleaned 3 times with 10% washing powder solution when step (1) gathers back laboratory explant
Afterwards, sterilized 15 seconds in 75% ethanol solution in being placed in superclean bench after being rinsed 20 minutes under running water, sterilized water
Rinse 2 times to be sterilized 15 minutes with after aseptic filter paper suck dry moisture, being placed in 0.1% mercuric chloride solution afterwards, after aseptic water washing 3 times
With being cut into the stem-segment with node of 1.0~1.5cm or so after aseptic filter paper suck dry moisture and be inoculated into adventitious bud induction culture base,
25 DEG C carry out full light culture induced synthesis adventitious bud by 25 days, and inductivity is 82.8%, and pollution rate is less than 20%.It is described not
Normal bud inducing culture is：MS culture medium+1.0mg/L ZT (zeatin)+0.3mg/L NAA+25g/L sucrose+3.5g/L agar
+ 0.5g/L activated carbons, pH is 5.4.
(3) squamous subculture：The adventitious bud that step (2) induction is obtained is cut into and is about the stem section of 1~2cm and is inoculated into subculture
The subculture for being capable of achieving adventitious bud for 25 days is cultivated in culture medium, growth coefficient reaches 7 times.Described subculture medium is：MS is trained
Base+1.0mg/L 6-BA+0.5mg/L NAA+20g/L sucrose+3.5g/L agar+0.1g/L activated carbons are supported, pH is 5.4.
(4) culture of rootage：The adventitious bud for being about 2.0~3.0cm that step (3) subculture is obtained is cut and be inoculated with from base portion
Culture can realize taking root for test tube seedling for 25 days in root media, and rooting rate is 89.5%.Described root media is：
1/2MS+0.3mg/L 6-BA+0.3mg/L IBA+1.0mg/L NAA+15g/L sucrose+3.0g/L agar, pH is 5.4.
(5) transplant：By the good test tube seedling of step (4) root growth in the natural light lower refining seedling 1 day in greenhouse, root is cleaned
Culture medium transplant in volume ratio be 1:Seedling is cultivated in 1 fertile soil, bark mixed-matrix and obtains final product seedling, after transplanting 30 days into
Motility rate is 91.8%.
The condition of culture of above-mentioned steps (3)~(4) is：Cultivation temperature is 25 DEG C, and intensity of illumination is 2000lx, light application time
It is 10 hours/day.
Embodiment two
(1) explant collection：Robust growth is chosen in the wild, the stem-segment with node of disease-free blue or green pod leaf plant is explant
Body, carries out water conservation moisturizing treatment and takes back laboratory in time immediately after collection.
(2) adventitious bud inducing：First cleaned 4 times with 10% washing powder solution when step (1) gathers back laboratory explant
Afterwards, sterilized 23 seconds in 75% ethanol solution in being placed in superclean bench after being rinsed 25 minutes under running water, sterilized water
Rinse 2 times to be sterilized 24 minutes with after aseptic filter paper suck dry moisture, being placed in 0.1% mercuric chloride solution afterwards, after aseptic water washing 4 times
With being cut into the stem-segment with node of 1.0~1.5cm or so after aseptic filter paper suck dry moisture and be inoculated into adventitious bud induction culture base,
27 DEG C carry out full light culture induced synthesis adventitious bud by 27 days, and inductivity is 81.3%, and pollution rate is less than 15%.It is described not
Normal bud inducing culture is：MS culture medium+0.8mg/L ZT (zeatin)+0.2mg/L NAA+23g/L sucrose+3.7g/L agar
+ 0.7g/L activated carbons, pH is 5.6.
(3) squamous subculture：The adventitious bud that step (2) induction is obtained is cut into and is about the stem section of 1~2cm and is inoculated into subculture
The subculture for being capable of achieving adventitious bud for 27 days is cultivated in culture medium, growth coefficient reaches 6 times.Described subculture medium is：MS is trained
Base+0.7mg/L 6-BA+0.3mg/L NAA+25g/L sucrose+3.7g/L agar+0.3g/L activated carbons are supported, pH is 5.6.
(4) culture of rootage：The adventitious bud for being about 2.0~3.0cm that step (3) subculture is obtained is cut and be inoculated with from base portion
Culture can realize taking root for test tube seedling for 28 days in root media, and rooting rate is 90%.Described root media is：1/
2MS+0.2mg/L 6-BA+0.2mg/L IBA+0.8mg/L NAA+17g/L sucrose+3.5g/L agar, pH is 5.6.
(5) transplant：By the good test tube seedling of step (4) root growth in the natural light lower refining seedling 2 days in greenhouse, root is cleaned
Culture medium transplant in volume ratio be 1:Seedling is cultivated in 1 fertile soil, bark mixed-matrix and obtains final product seedling, after transplanting 30 days into
Motility rate is 93%.The condition of culture of above-mentioned steps (3)~(4) is：Cultivation temperature is 26 DEG C, and intensity of illumination is 2700lx, illumination
Time is 11 hours/day.
Embodiment three
(1) explant collection：Robust growth is chosen in the wild, the stem-segment with node of disease-free blue or green pod leaf plant is explant
Body, carries out water conservation moisturizing treatment and takes back laboratory in time immediately after collection.
(2) adventitious bud inducing：First cleaned 5 times with 10% washing powder solution when step (1) gathers back laboratory explant
Afterwards, sterilized 30 seconds in 75% ethanol solution in being placed in superclean bench after being rinsed 30 minutes under running water, sterilized water
Rinse 3 times to be sterilized 20 minutes with after aseptic filter paper suck dry moisture, being placed in 0.1% mercuric chloride solution afterwards, after aseptic water washing 5 times
With being cut into the stem-segment with node of 1.0~1.5cm or so after aseptic filter paper suck dry moisture and be inoculated into adventitious bud induction culture base,
28 DEG C carry out full light culture induced synthesis adventitious bud by 30 days, and inductivity is 80.9%, and pollution rate is 9.2%.It is described not
Normal bud inducing culture is：MS culture medium+0.5mg/L ZT (zeatin)+0.1mg/L NAA+30g/L sucrose+4.0g/L agar
+ 1.0g/L activated carbons, pH is 5.8.
(3) squamous subculture：The adventitious bud that step (2) induction is obtained is cut into and is about the stem section of 1~2cm and is inoculated into subculture
The subculture for being capable of achieving adventitious bud for 30 days is cultivated in culture medium, growth coefficient reaches 5 times.Described subculture medium is：MS is trained
Base+0.5mg/L 6-BA+0.1mg/L NAA+30g/L sucrose+4.0g/L agar+0.5g/L activated carbons are supported, pH is 5.8.
(4) culture of rootage：The adventitious bud for being about 2.0~3.0cm that step (3) subculture is obtained is cut and be inoculated with from base portion
Culture can realize taking root for test tube seedling for 30 days in root media, and rooting rate is 86.1%.Described root media is：
1/2MS+0.1mg/L 6-BA+0.1mg/L IBA+0.5mg/L NAA+20g/L sucrose+4.0g/L agar, pH is 5.8.
(5) transplant：By the good test tube seedling of step (4) root growth in the natural light lower refining seedling 3 days in greenhouse, root is cleaned
Culture medium transplant in volume ratio be 1:Seedling is cultivated in 1 fertile soil, bark mixed-matrix and obtains final product seedling, after transplanting 30 days into
Motility rate is 93.7%.
The condition of culture of above-mentioned steps (3)~(4) is：Cultivation temperature is 28 DEG C, and intensity of illumination is 2500lx, light application time
It is 12 hours/day
Above-described embodiment is the present invention preferably implementation method, but embodiments of the present invention are not by above-described embodiment
Limitation, it is other it is any without departing from Spirit Essence of the invention and the change, modification, replacement made under principle, combine, simplification,
Equivalent substitute mode is should be, is included within protection scope of the present invention.
Claims (9)
1. the rapid propagation method of a kind of the Miaos medicinal materials green grass or young crops pod leaf, it is characterised in that：Including the following steps for carrying out successively：Obtain
The explant of blue or green pod leaf, adventitious bud inducing, the squamous subculture of adventitious bud, the culture of rootage of adventitious bud, test tube transplantation of seedlings；
The inducing culture of described adventitious bud inducing includes：MS culture mediums, 0.5~1.0mg/L ZT, 0.1~0.3mg/L
NAA, 25~30g/L sucrose, 3.5~4.0g/L agar, 0.5~1.0g/L activated carbons, pH are 5.4~5.8.
2. the rapid propagation method of the Miaos medicinal materials according to claim 1 green grass or young crops pod leaf, it is characterised in that：Described adventitious bud
The method of induction is：By the explant of blue or green pod leaf is cleaned disinfect after be cut into the stem-segment with node of 1.0~1.5cm and be inoculated into
In inducing culture, induced synthesis adventitious bud by carrying out full light culture 25~30 days at 25~28 DEG C.
3. the rapid propagation method of the Miaos medicinal materials according to claim 1 green grass or young crops pod leaf, it is characterised in that：The subculture of adventitious bud
Culture subculture medium used includes：MS culture mediums, 0.5~1.0mg/L 6-BA, 0.1~0.5mg/L NAA, 20~30g/
L sucrose, 3.5~4.0g/L agar, 0.1~0.5g/L activated carbons, pH are 5.4~5.8.
4. the rapid propagation method of the Miaos medicinal materials according to claim 3 green grass or young crops pod leaf, it is characterised in that：Described adventitious bud
The method of squamous subculture be specially：The adventitious bud obtained by adventitious bud inducing is cut into the stem section of 1~2cm long and is inoculated into
The subculture for being capable of achieving adventitious bud for 25~30 days is cultivated in subculture medium.
5. the rapid propagation method of the Miaos medicinal materials according to claim 1 green grass or young crops pod leaf, it is characterised in that：Adventitious bud is taken root
Culture root media used includes：1/2MS, 0.1~0.3mg/L 6-BA, 0.1~0.3mg/L IBA, 0.5~1.0mg/
L NAA, 15~20g/L sucrose, 3.0~4.0g/L agar, pH are 5.4~5.8.
6. the rapid propagation method of the Miaos medicinal materials according to claim 5 green grass or young crops pod leaf, it is characterised in that：Described adventitious bud
The method of culture of rootage be：The adventitious bud of the 2.0~3.0cm of length that will be obtained by the squamous subculture of adventitious bud cuts from base portion
And culture can realize taking root for test tube seedling in 25~30 days in being inoculated into root media.
7. the rapid propagation method of the Miaos medicinal materials according to claim 1 green grass or young crops pod leaf, it is characterised in that：Described test tube seedling
The method of transplanting is specially：The well-grown test tube seedling that will be obtained by the culture of rootage of adventitious bud is under the natural light in greenhouse
Hardening 1~3 day, it is 1 that the culture medium of clean root is transplanted in volume ratio:Seedling is cultivated in 1 fertile soil, bark mixed-matrix is
Obtain seedling.
8. according to the rapid propagation method of any described the Miaos medicinal materials green grass or young crops pod leaf of claim 1 to 7, it is characterised in that：It is described
Adventitious bud inducing, the condition of culture of the squamous subculture of adventitious bud is：Cultivation temperature is 25~28 DEG C, and intensity of illumination is 2000
~2500lx, light application time is 10~12 hours/day.
9. according to the rapid propagation method of any described the Miaos medicinal materials green grass or young crops pod leaf of claim 1 to 7, it is characterised in that：Obtain
The specific method of the explant of blue or green pod leaf is：Choosing robust growth, the stem-segment with node of disease-free blue or green pod leaf plant in the wild is
Explant, carries out water conservation moisturizing treatment immediately after collection.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102599061A (en) * | 2012-03-29 | 2012-07-25 | 常熟市海虞茶叶有限公司 | Method for rapid propagating tissue of cornus alba |
| CN104719134A (en) * | 2014-10-08 | 2015-06-24 | 江苏碧云天农林科技有限公司 | Dendrobenthemia japonica tissue culture method |
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2017
- 2017-03-13 CN CN201710147110.4A patent/CN106879466B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102599061A (en) * | 2012-03-29 | 2012-07-25 | 常熟市海虞茶叶有限公司 | Method for rapid propagating tissue of cornus alba |
| CN104719134A (en) * | 2014-10-08 | 2015-06-24 | 江苏碧云天农林科技有限公司 | Dendrobenthemia japonica tissue culture method |
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