CN106305429A - In-vitro rapid propagation technology for prinsepia sinensis - Google Patents
In-vitro rapid propagation technology for prinsepia sinensis Download PDFInfo
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- CN106305429A CN106305429A CN201610814396.2A CN201610814396A CN106305429A CN 106305429 A CN106305429 A CN 106305429A CN 201610814396 A CN201610814396 A CN 201610814396A CN 106305429 A CN106305429 A CN 106305429A
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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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- Developmental Biology & Embryology (AREA)
- Engineering & Computer Science (AREA)
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- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Cultivation Of Plants (AREA)
Abstract
The invention belongs to the field of plant tissue culture and provides an in-vitro rapid propagation technology for prinsepia sinensis. The in-vitro rapid propagation technology includes that tender stem segments of the prinsepia sinensis are taken as explants and MS as a minimum medium, plant growth regulator 6-BA and IBA are added to induce axillary buds to sprout and grow, GA3 is added in the process of transgenerational proliferation to promote cluster buds to grow high, and a great deal of inherited-consistent regeneration plants can be obtained within a short period via root induction and acclimatization and transplant. Technical support is provided for rapid propagation of fine breed, and the technology is of great practical significance in speeding comprehensive development and utilization of the prinsepia sinensis.
Description
Technical field
The invention belongs to field of plant tissue culture, be specifically related to the rapid propagation in vitro technology of a kind of Prinsepia sinensis.
Background technology
Prinsepia sinensisPrinsepia sinensis(Oliv.) Oliv.ex Bean. has another name called Liaoning Folium Prinsepiae Utilis, shoulder pole
Beard, northeast Rui core, for Rosaceae (Rosaceae) Prinsepia Royle machaka.Being distributed in China northeast, NORTHERN KOREA also has
Distribution.These seeds are cold-resistant, and leaf bud is sprouted early, bloom early, the full simple and elegant delicate fragrance of branch Hemerocallis citrina Baroni, and axillary shoot stings upright or bending, and fruit is red
Moisten exquisitely carved, be that rising the seeing in the north is spent, appreciated spiny plant really.
The fruit of Prinsepia sinensis is sour-sweet and succulence, containing several amino acids, vitamin, various trace elements and saccharide,
Can eat or be processed into the natural green foods such as preserved fruit, fruit juice, fruit jam raw, also can make wine.Pit decorative pattern is the most embedding, oblate, can add
Work becomes artwork ornaments；Kernel is used as medicine liver heat removing effect tomorrow, is usually used in treating the oculopathy such as eye conjunctivitis, corneal nebula, is doctor
Medicine products that are in short supply, supply falls short of demand.
Prinsepia sinensis has the highest Landscape Application and is worth and economic worth, but current existing wild resource is little, because of
It is imperative that this cultivates a large amount of high quality seedlings.Prinsepia sinensis is mainly bred by the method for sowing and cuttage at present.But its kind
Sub-pit is hard, and seed propagation is relatively difficult, and seedling needs more than 15 years ability solid.Cottage propagation is originated by cutting
Limiting with season, the speed of growth is slow, there is the problem that survival rate is low, constrains popularizing planting and the application of Prinsepia sinensis.Cause
This seems the most urgent to the research of its tissue culture technique.Therefore, it is necessary to set up the rapid propagation in vitro of a kind of Prinsepia sinensis
Technology, provides technical support and guarantee for its breeding fast breeding, develops further for it and lays the foundation.
Summary of the invention
It is an object of the invention to provide the rapid propagation in vitro technology of a kind of Prinsepia sinensis, realize scale breeding.Pass through
Outer implant selects and the process such as sterilization, startup inducing culture, subculture multiplication cultivation, root induction cultivation, acclimatization and transplants achieves
Prinsepia sinensis rapid propagation in vitro.
The present invention provides a kind of Prinsepia sinensis rapid propagation in vitro technology, comprises the following steps:
(1) sterilization of outer implant and Primary culture: the healthy and strong edible tender branch without pest and disease damage of growth selection is outer implant, first with flowing
Water rinses 30min, cuts off the blade on sprout, stays 2～3 mm petioles, then with 10%Ca (CLO)2Sterilizing 20 min, aseptic
Water rinses 3 times.Twig is trimmed to 1cm left and right belt leaf stem section be inoculated in startup inducing culture on, be placed in illumination every day 10～
12h, intensity of illumination is 15001x, and cultivation temperature is cultivated under the conditions of being 23～25 DEG C, until induction axillary bud sprouting growth；
(2) subculture multiplication is cultivated: the sprout of axillary bud sprouting elongation growth proceeds to carry out on proliferated culture medium subculture multiplication cultivation,
Being placed in illumination every day 12 hours, intensity of illumination is 15001x, and cultivation temperature is cultivated under the conditions of being 23～25 DEG C, until axillary bud sprouting
Or formation Multiple Buds, subculture cycle is 35d；
(3) root induction is cultivated: what height was about 3cm robust growth is inoculated on root media without root that carrying out takes root lures
Leading, be placed in illumination every day 12 hours, intensity of illumination is 2000～25001x, cultivation temperature cultivate under the conditions of being 23～25 DEG C to
Take root；
(4) acclimatization and transplants: after Prinsepia sinensis cultivates 40～45d in root media, selects robust growth, well developed root system
Tissue cultured seedling, first open sealed membrane and be placed in natural lighting lower refining seedling 3～5d, then clean tissue cultured seedling root residual with clear water
Culture medium, then soaks 20～30min with 800 times of carbendazim solutions, then be transplanted to the perlite of equal-volume ratio sterilized and
In Vermiculitum mixed-matrix.
Primary culture base described in above-mentioned steps (1) is MS+1.0mg/L 6-BA (6 benzyl aminoadenine)+0.1mg/L
IBA(indolebutyric acid)+30g/L sucrose+7g/L agar, pH value is 5.8.Through the inducing culture of about 20d, axillary bud sprouting is taken out
Branch, branch long about 3cm during 35d.
Proliferated culture medium described in above-mentioned steps (2) is MS+1.0mg/L 6-BA+0.2mg/L IBA+1.0～2.0mg/
LGA3+ 30g/L sucrose+7g/L agar, pH value is 5.8, and growth coefficient reaches more than 6.
Root media described in above-mentioned steps (3) is 1/2B5+ 0.5mg/L IBA+1.5mg/L NAA+15g/L sucrose+
7g/L agar, pH value is 5.8.About 20d starts to take root, and about 45d produces 5～8 sturdy adventitious roots, root length average out to
2cm, rooting rate reaches 85%.
Substrate 0.3% potassium permanganate solution is drenched sterilization before transplanting by above-mentioned steps (4), use the most again clear water shower 3～
5 times.Transplant early stage covered with plastic film moisturizing and suitably shelter from heat or light with shading screen, spraying every day 1 time, sprayed more than 800 times every 5 days
Bacterium spirit germicidal solution, transplanting the later stage is gradually lowered humidity, increases illumination.Transplanting the long root that makes new advances of about 15d, transplanting survival rate reaches
More than 85%.Ensure that humiture balance and lighting requirements are that transplanting is the most crucial.
Beneficial effects of the present invention
Yet there are no the relevant report of Prinsepia sinensis rapid propagation in vitro at present both at home and abroad, the present invention uses plant tissue culture technique,
By induction axillary bud sprouting and the approach of formation Multiple Buds, a large amount of nursery stock can be bred at short notice.The present invention has propagation system
The features such as number is high, transplanting survival rate is high, growth cycle is short, substantially increase the breeding efficiency of Prinsepia sinensis, beneficially batch production
Produce.
Detailed description of the invention
Following example are to further illustrate the present invention, are not limitations of the present invention.
It is embodied as
(1) sterilization of outer implant and Primary culture: the edible tender branch sprouted then without pest and disease damage that growth selection is healthy and strong or sprout
Clockwork spring is outer implant, first rinses 30min with flowing water, then cuts off the blade on sprout, stays 2～3 mm petioles, then uses 10%Ca
(CLO)2Sterilizing 20 min, rinsed with sterile water 3 times.Twig is cut into 1cm left and right belt leaf stem section and is inoculated in startup inducing culture
On, it being placed in illumination every day 10～12h, intensity of illumination is 15001x, and cultivation temperature is cultivated under the conditions of being 23～25 DEG C.Used opens
Dynamic culture medium is MS+1.0mg/L 6-BA+0.1～0.3mg/L IBA+30g/L sucrose+7g/L agar, and pH value is 5.8.Pass through
The inducing culture of 20d, axillary bud sprouting branches out, 30～35d long about the 3cm of branch.
(2) subculture multiplication is cultivated: the sprout of axillary bud sprouting elongation growth proceeds to carry out on proliferated culture medium subculture multiplication
Cultivating, be placed in illumination every day 12 hours, intensity of illumination is 15001x, and cultivation temperature is cultivated under the conditions of being 23～25 DEG C.Propagation training
Foster base is MS+1.0mg/L 6-BA+0.2mg/L IBA+1.0～2.0mg/LGA3+ 30g/L sucrose+7g/L agar, pH value is
5.8.In initial Subculture, axillary bud sprouting and Seedling height are fast, repeatedly after successive transfer culture, are commonly formed Multiple Buds, increase
Growing coefficient and be more than 6, subculture cycle is 35d.
(3) root induction is cultivated: height is about being inoculated on root media without root of 3cm robust growth and gives birth to
Root induction, is placed in illumination every day 12 hours, and intensity of illumination is 2000～25001x, and cultivation temperature is trained under the conditions of being 23～25 DEG C
Support.Root media is 1/2B5+ 0.5mg/L IBA+0.5～1.5mg/L NAA+15g/L sucrose+7g/L agar, pH value is
5.8.When NAA concentration is 1.5 mg/L, cultivating about 20d and start to take root, about 45d produces 5～8 sturdy adventitious roots, root
Long average out to 2cm, rooting rate is 85%.And when NAA concentration is less than 1.5mg/L, rooting rate is low and rootage duration is long.
(4) acclimatization and transplants: after Prinsepia sinensis cultivates 45d in root media, selects robust growth, well developed root system
Tissue cultured seedling, first open sealed membrane and be placed in natural lighting lower refining seedling 3～5d, then clean the cultivation of root residual with clear water
Base, is transplanted in the substrate sterilized (V perlite: V Vermiculitum=1:1), ensures temperature and humidity conditions, meticulously manage and protect during transplanting,
Transplanting survival rate reaches more than 85%.
Claims (4)
1. the rapid propagation in vitro technology of a Prinsepia sinensis, it is characterised in that comprise the following steps:
(1) sterilization of outer implant and Primary culture: the healthy and strong edible tender branch sprouted then without pest and disease damage of growth selection is outward
Implant, first rinses 30min with flowing water, cuts off the blade on sprout, stays 2～3 mm petioles, then with 10%Ca (CLO)2Sterilizing
20 min, rinsed with sterile water 3 times, twig is trimmed to 1cm left and right belt leaf stem section and is inoculated on startup inducing culture, be placed in every
It illumination 10～12h, intensity of illumination is 15001x, and cultivation temperature is cultivated under the conditions of being 23～25 DEG C, until induction axillary bud growth；
(2) subculture multiplication is cultivated: the sprout of axillary bud sprouting elongation growth proceeds to carry out on proliferated culture medium subculture multiplication cultivation,
Being placed in illumination every day 12 hours, intensity of illumination is 15001x, and cultivation temperature is cultivated under the conditions of being 23～25 DEG C, until axillary bud sprouting
Or formation Multiple Buds, subculture cycle is 35d；
(3) root induction is cultivated: being inoculated on root media without root of robust growth that height is about 3cm taken root
Induction, is placed in illumination every day 12 hours, and intensity of illumination is 2000～25001x, and cultivation temperature is cultivated under the conditions of being 23～25 DEG C
To taking root；
(4) acclimatization and transplants: after Prinsepia sinensis cultivates 40～45d in root media, selects robust growth, well developed root system
Tissue cultured seedling, first open sealed membrane and be placed in natural lighting lower refining seedling 3～5d, then clean the cultivation of root residual with clear water
Base, is transplanted in the perlite of equal-volume ratio sterilized and Vermiculitum mixed-matrix.
The rapid propagation in vitro technology of a kind of Prinsepia sinensis the most according to claim 1, it is characterised in that described in step (1)
Initial culture base is: MS+1.0mg/L 6-BA (6 benzyl aminoadenine)+0.1mg/L IBA(indolebutyric acid)+30g/L sugarcane
Sugar+7g/L agar, pH value is 5.8.
The rapid propagation in vitro technology of a kind of Prinsepia sinensis the most according to claim 1, it is characterised in that described in step (2)
Proliferated culture medium is MS+1.0mg/L 6-BA+0.2mg/L IBA+1.0～2.0mg/LGA3(gibberellins)+30g/L sucrose+7g/
L agar, pH value is 5.8.
The rapid propagation in vitro technology of a kind of Prinsepia sinensis the most according to claim 1, it is characterised in that described in step (3)
Root media is 1/2B5+ 0.5mg/L IBA+1.5mg/L NAA(naphthalene acetic acid)+15g/L sucrose+7g/L agar, pH value is
5.8。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610814396.2A CN106305429B (en) | 2016-09-12 | 2016-09-12 | A kind of rapid propagation in vitro method of Prinsepia sinensis |
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| CN201610814396.2A CN106305429B (en) | 2016-09-12 | 2016-09-12 | A kind of rapid propagation in vitro method of Prinsepia sinensis |
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| CN106305429A true CN106305429A (en) | 2017-01-11 |
| CN106305429B CN106305429B (en) | 2018-04-20 |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005012507A1 (en) * | 2003-07-25 | 2005-02-10 | The University Of Melbourne | Production of plant secondary metabolites using adsorption and elicitation in cell suspension culture |
| CN1657090A (en) * | 2004-02-18 | 2005-08-24 | 丁庆 | Extract of prinsepia wood and its bioactivity function |
| CN104737908A (en) * | 2015-03-06 | 2015-07-01 | 朱远星 | Cerasus humilis tissue culture rapid propagation method |
-
2016
- 2016-09-12 CN CN201610814396.2A patent/CN106305429B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005012507A1 (en) * | 2003-07-25 | 2005-02-10 | The University Of Melbourne | Production of plant secondary metabolites using adsorption and elicitation in cell suspension culture |
| CN1657090A (en) * | 2004-02-18 | 2005-08-24 | 丁庆 | Extract of prinsepia wood and its bioactivity function |
| CN104737908A (en) * | 2015-03-06 | 2015-07-01 | 朱远星 | Cerasus humilis tissue culture rapid propagation method |
Non-Patent Citations (2)
| Title |
|---|
| BHAGAT S, ET AL.: "STUDIES ON DETERMINATION OF OPTIMUM SOWING METHOD OF PRINSEPIAUTILIS ROYLE SEED IN THE FIELD", 《INDIAN FORESTER》 * |
| 曾妮等: "青刺果离体快速繁殖", 《植物生理学通讯》 * |
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Granted publication date: 20180420 Termination date: 20200912 |