CN104542300B

CN104542300B - The culture medium in osmund cultured in vitro each stage

Info

Publication number: CN104542300B
Application number: CN201510042794.2A
Authority: CN
Inventors: 邓坦; 周逊; 王坤兰; 韩芳; 谢秋桃; 顾婵; 韦唯
Original assignee: Zunyi Normal University
Current assignee: Zunyi Normal University
Priority date: 2015-01-28
Filing date: 2015-01-28
Publication date: 2016-08-24
Anticipated expiration: 2035-01-28
Also published as: CN104542300A

Abstract

A kind of culture medium in osmund cultured in vitro each stage, the culture medium in each stage is as follows: 1. the preferable culture medium of inducing spore sprouting is: 1,/2M,S+2 3% sucrose agar culture medium, 2. original foliage (gametophyte) proliferation culture medium formula MS+0.2 0.6mg/L KT+1.0 1.5mg/L IBA agar medium.3. induction original foliage fertilization sprouting sporinite formula is: MS fluid nutrient medium, and 4. the preferable culture medium of sporinite propagation is: MS+0.2 0.6mg/L NAA+0.5 1.0mg/L 6 BA fluid nutrient medium.Beneficial effect: providing the culture medium prescription breeding each stage, be favorably improved the fertility of osmund, the simplest and the most direct cultural method is advantageously implemented the large-scale production of osmund, no matter for enterprise or peasant, has sizable economic benefit and social benefit.

Description

The culture medium in osmund cultured in vitro each stage

Technical field

The present invention relates to the tissue culture technique of pteridophyte, particularly relate to the culture medium in osmund cultured in vitro each stage.

Background technology

Osmund (Osmunda japonica Thunb) belongs to Pteridophyta, true fern, original thick capsule Filicopsida, osmund Mesh, Osmundaceae, a kind of herbaceos perennial of Osmunda, it is a kind of wild vegetables and a kind of rare traditional Chinese medicine.Border, Guizhou Nei Ge county has osmund (common vetch dish) to be distributed, and common people pluck wild osmund annual spring is processed into Dried Osmund sale or edible,.Purple Containing flavonoids, lactone, ketosteroid, polysaccharide, tannin and Amino acids in beanstalk.Dried Osmund is rich in protein, amino Acid, cellulose, carbohydrate, and the nutritional labeling such as potassium, silicon, magnesium, phosphorus, iron, zinc, nutritional quality is excellent, can with gold dried vegetable, The famous and precious mountain delicacy food such as auricularia auriculajudae, dictyophora phalloidea matches in excellence or beauty, and is the famous and precious wild vegetables of one on the ground such as stub Japan of current China, Korea S.Osmund Rhizome and petiole residue can clearing heat and detoxicating, stop blooding desinsection, slightly poisonous.Utilize osmund for raw material effective component extracting for curing Pharmaceutical products and exploitation health food, meet the developing direction of world today's food science and preventive medicine.But due to osmund certainly So reproductive capacity is low, and substantial amounts of digging wild fern resource is as mother's kind, and the protection for water and soil conservation and wild resource is unfavorable , limit the large-scale production of common vetch dish, therefore, carrying out tissue cultures with sporinite for material has in osmund large-scale production The biggest development and application prospect.

Osmund (common vetch dish), in its history of life, sporinite to be experienced and gametophyte two eposides, its gametophyte sexual intercourse Needing water as medium in Process of Insemination from generation to generation, be difficult to succeed by conventional in-vitro culture method, the present invention is according to fern This feature of class plant, explores the combination formula of a set of osmund (common vetch dish) cultured in vitro.

Summary of the invention

It is an object of the invention to provide the culture medium in a kind of osmund cultured in vitro each stage, carry out group with sporinite for material Knit cultivation, overcome osmund natural propagation power low, limit the large-scale production of common vetch dish, provide technology bar for osmund large-scale production Part.

The culture medium in concrete each stage is as follows:

1) inducing spore body is sprouted and is formed original foliage culture medium prescription:

1/2MS+2-3% sucrose agar culture medium.

2) original foliage (gametophyte) proliferation culture medium formula:

MS+0.2-0.6mg/L KT+1.0-1.5mg/L IBA agar medium.

3) induction original foliage fertilization sprouting sporinite formula:

MS fluid nutrient medium.

4) sporinite proliferation culture medium formula:

MS+0.2-0.6mg/L NAA+0.5-1.0mg/L 6-BA fluid nutrient medium.

The beneficial effect of the technical program: the culture medium prescription breeding each stage is provided, is favorably improved the breeding of osmund Ability, the simplest and the most direct cultural method is advantageously implemented the large-scale production of osmund, no matter for enterprise or peasant, has phase When big economic benefit and social benefit.

Detailed description of the invention

Illustrated by specific embodiment below

Gathering differing maturity plant number strain, the fertile spike of the osmund of clip differing maturity, is used laundry respectively Powder soaks 5min, washes away surface dirt, then rinses 30min with running water, under aseptic condition, by the alcohol disinfecting 60s of 75%, nothing Bacterium water rinses 3 times, then the mercuric chloride solution sterilizing 8min with 0.1%, aseptic water washing 4～5 times, then blots table with aseptic blotting paper Face moisture, is cut into the segment of 0.5cm length, is then inoculated in spore germination culture medium.

Inducing spore body is sprouted and is formed original foliage

Inducing spore body is sprouted and is formed the culture medium of original foliage with MS culture medium as minimal medium, MS is divided into 1/4,1/2, 1 three level concentration, sucrose is divided into 2%, 3% two horizontal (see Table 1), and inoculation material osmund fertile spike maturity is divided into light green Tri-levels of the Lao Miao of the seedling of look, bottle-green strong sprout and brown, are designated as maturity I, maturity II, maturity III respectively, Access the fertile spike section of 1 0.5cm length, put into culturing room and cultivate.Observe statistics survival rate, and be combined into motility rate and growth shape State, filters out preferable culture medium prescription.

The culture medium prescription that table 1 inducing spore body is sprouted

The original foliage that spore germination is formed is inoculated in the culture medium of MS+0.5mg/L KT+1.5mg/L IBA, puts into Culturing room carries out original foliage Multiplying culture.

Sporinite is sprouted in original foliage induction fertilization

The original foliage bred is taken out and puts into equipped with in the aseptic triangular flask of MS fluid nutrient medium, shaking table shakes respectively Swing 5min, 10min, 30min, take appropriate original foliage the most respectively and access equipped with in the sterile culture flask of cotton, then pour into The MS Liquid Culture of 20ml, based in sterile culture flask, is put into culturing room and is cultivated.Each process inoculates 20 bottles, repeats for totally three times. Gametophytic induction and developmental state in each blake bottle is recorded respectively, to filter out induction when one and a half months and three first quarter moons Optimal duration of oscillation and the incubation time of sporinite is sprouted in original foliage fertilization.

The propagation of sporinite

The culture medium prescription of sporinite propagation is that MS+NAA+6-BA, NAA are divided into 0mg/L, 0.1mg/L, 0.5mg/L tri- Level, 6-BA is divided into tri-levels of 0.1mg/L, 0.5mg/L, 1.0mg/L, totally 9 process (being shown in Table 2), each process 15 bottles, often Bottle 4 young plants, repeat for three times.Put into culturing room to cultivate.Observe statistics, be calculated as motility rate, number of emerging, in conjunction with growth conditions, screening Go out sporinite and breed preferable culture medium prescription.

The culture medium prescription of table 2 sporinite breeding

Condition of culture

Culturing room's temperature controls at 25 ± 1 DEG C, and light application time is 12h/d, intensity of illumination 2500lx.

Conclusion

The relatively MS culture medium of different ratio, different sucrose, hormone concentration affects situation to osmund cultured in vitro, To filter out the culture medium prescription in applicable osmund seedling proliferation expansion numerous each stage.Result shows: inducing spore sprouts preferably training Foster base is: 1/2MS+2-3% sucrose agar culture medium, and sugar concentration is relatively big to Spore Germination, relatively closes in the range of 2%～3% Suitable, less than 2% with exceed 4%, spore germination rate substantially reduces.There are some researches show, in the gametophytic Fiber differentiation of osmund is tested, Routine is that the original foliage after breeding takes out, and is placed in beaker and smashs the stirring that adds water to pieces, then is inoculated in subculture medium equably On (solid medium), the original foliage incision smashed to pieces can grow again the original foliage made new advances.This experiment uses Liquid Culture Base, its operating process is simpler and more direct compared with for solid medium.Sporinite breeds preferable culture medium: MS+0.2- 0.6mg/L NAA+0.5-1.0mg/L 6-BA fluid nutrient medium.Pertinent literature is separately had to record: culture medium 1/8MS, 1/2MS, MS+ 1.0mg/L NAA+0.5mg/L 6-BA, can promote that sporinite is taken root.This conclusion together confirms with the result of the present invention, NAA/ When 6-BA is certain ratio, it is favourable to plant growth, when the value of NAA/6-BA is 1/2, breeds most beneficial for gametophyte； When the value of NAA/6-BA is 2, take root most beneficial for sporinite.

Claims

1. the culture medium in osmund cultured in vitro each stage, it is characterised in that the culture medium in concrete each stage is as follows:

1/2MS+2-3% sucrose agar culture medium；

2) original foliage i.e. gametophyte proliferation culture medium formula:

MS+0.2-0.6mg/L KT+1.0-1.5mg/L IBA agar medium；

3) induction original foliage fertilization sprouting sporinite formula:

MS fluid nutrient medium；

4) sporinite proliferation culture medium formula:

MS+0.2-0.6mg/L NAA+0.5-1.0mg/L 6-BA fluid nutrient medium.