CN104396758A

CN104396758A - Dendrobium officinale embryoid culture process

Info

Publication number: CN104396758A
Application number: CN201410748277.2A
Authority: CN
Inventors: 张�杰
Original assignee: Individual
Current assignee: Individual
Priority date: 2014-12-10
Filing date: 2014-12-10
Publication date: 2015-03-11

Abstract

The invention relates to a dendrobium officinale embryoid culture process which comprises the following steps: adopting a wild dendrobium adult plant stem segment to induce an embryoid, carrying out initial dendrobium embryoid induction on a culture medium filled with a proper amount of carbon sources, fruit and vegetable juice, plant growth regulators and agar powder under proper temperature, physiological activity illumination intensity and suitable illumination time and carrying out amplification at proper time, performing transferring into the culture medium after the completion of amplification to carry out suspension culture and finally obtaining an amplification system which has short production period and high polysaccharide content, has a specific biological growth rate reaching 8-9.5 milligram/gram/day and can be amplified to above 10 tons/year and keep stable every year.

Description

Dendrobium nobile embryoid culture technology

Technical field

The invention belongs to plant tissue field of engineering technology.The present invention relates to a Plant Biotechnology cloning engineering, particularly for picture this medical value of dendrobium candidum very high but poor growth, endangered species that output is extremely low, very harsh to requirement for environmental conditions.

Background technology

Along with the continuous degeneration developing the continuous growth with population, ecotope rapidly of urbanization process, make many rare traditional Chinese medicine resource exhaustions and be on the point of to go out.Particularly for picture this medical value of dendrobium candidum very high but poor growth, endangered species that output is extremely low, very harsh to requirement for environmental conditions, its social benefit is very large.

The stem of noble dendrobium one medicine, as the term suggests, be specially refer to the saxicolous stem of noble dendrobium.As for the Dendrobium Sw on trees of growing nonparasitically upon another plant, ancient times, book on Chinese herbal medicine was also on the books, was referred to as wooden dry measure used in former times.Collective Notes to the Canon of Materia medica day: " person's name wood dry measure used in former times on raw robur, its stem shape is grown up and look shallow.The modern peace that begins also goes out wooden dry measure used in former times, to virtual length, does not enter ball and falls apart "." Bencao Tujing " day: " only person's victory on raw stone.Also have person on raw oak, name wood dry measure used in former times, can't bear to use." the current wooden dry measure used in former times be extensively incubated on wood chip, the property of medicine is very micro-, and large area land occupation.Although from eighties of last century eighties, people attempt with seminal propagation, means such as artificial cultivation or by tissue cultures, and the former stem ball of induction seed increases plant, then carries out the methods such as artificial introducing and planting, expands the production of dendrobium candidum.But effect is very micro-so far.And the method utilizing seed sprouting to increase is easy to cause mutation, very large threat is caused to the purity of stem of noble dendrobium species.

The present invention is according to two of higher plant cell totipotencies: plant cell has the totipotency that the totipotency that is divided into whole plant and plant cell can synthesize the medicinal effective ingredient of primary plant.According to such theoretical foundation, for these concrete species of dendrobium candidum, invent a whole set of and directly induced embryoid from dendrobium candidum primary plant strain stem section sleeping bud, ensure that from source " genuineness " of medicinal material.

Utilize dendrobium nobile embryoid suspension culture techniques technique of the present invention, its growth rate reaches 8-9.5 milligram/gram sky; Be equivalent to more than 1300 times of self-sow speed.Within every 7 days, results once must do rate more than 10%.Due to vegetative propagation, there is not the danger of transmutation of species, its genetic stability is reliable.By implementing technical matters of the present invention, the biological culture scale of dendrobium candidum being transformed from experimental level to factorial praluction scale and provides material guarantee.

Summary of the invention

The invention discloses the segment directly cutting band joint from stem of noble dendrobium band dormancy leaf stem section, be inserted into interpolation carbon source, appropriate Juice, plant growth regulator, on the plant tissue culture media of agar powder, at suitable PH, under temperature and light shines intensity, through cultivating after a while, when initial embryoid grows to suitable size, in time embryoid ball is separated explant, be placed on identical above-mentioned medium and increase.Being transferred to above-mentioned medium after amplification removes in the liquid nutrient medium of agar powder, carries out suspension and cultivates, and every bottle, by suitable rate of vaccination inoculation, in the growth of revolution shaking table, is carried out subculture depending on its upgrowth situation; After repeatedly secondary subculture; Enter screening sequence, from culture, constantly pick out light green color is spherical shape, the embryoid that growth is fine and close, amplification rate is fast, pass through step sizing, finally reach every 7 days results once, polyoses content reaches more than 10%, than biological growth rate 8 ~ 9.5 milligrams of/gram of skies, and the amplification system keeping the anniversary stable.

Owing to adopting clone technology, its genetic stability, detect completely identical with former plant through DNA.Dry powder that can increase, that meet the medicinal cost-effectively of the primary plant of the stem of noble dendrobium can be produced by technical matters of the present invention, expand apparent availability, meet the needs of people.

Case study on implementation

In conjunction with case study on implementation, the present invention is done to set forth, but be not restriction limitation of the present invention.

(see photo) is cultivated in the suspension of embodiment 1. dendrobium nobile embryoid

1. dendrobium candidum explant collection, sterilizing: take tender stem segments as explant, disinfect through explant, be inoculated in protocorm induction medium, through induction after a while, explant expands and produces protocorm, is transferred in proliferated culture medium by the protocorm of induced synthesis and cultivates.Minimal medium: MS medium, on the solid culture medium of carbon source, fruit juice, agar powder, PH5.8.Condition of culture: temperature (25 ± 5) DEG C, light intensity is 20001x, light application time 12-20h/ days.

2. the inducing culture of initial embryoid: above-mentioned solid culture medium has been inoculated explant triangular flask and has been placed on 25 DEG C ± 5, intensity of illumination is 20001x, light application time 12-20h/ days, and after a couple of days continuous culture, initial embryoid grows on stem section sleeping bud.By the time during appropriate size, carefully by the explant that embryoid is peeled off, and be transferred on the above-mentioned identical solid culture medium of newly joining, in each triangular flask, place several embryoid ball, be beneficial to form " conditioned medium ", promote the propagation of embryoid ball further.

3. embryoid suspension medium and screening sequence:

A. suspension medium; Above-mentioned solid culture medium changes over liquid nutrient medium, with condition sterilizing above-mentioned;

B. embryoid suspends and cultivates: after waiting the embryoid growing up to q.s in above-mentioned solid culture medium, by embryoid by certain rate of vaccination, is inoculated into 200 milliliters of triangular flasks; Triangular flask aluminium platinum paper seals, and is put on 90 ~ 120 revs/min of swinging shaking tables, and 25 DEG C ± 5, light intensity is 20001x, light application time 12-20h/ days, carries out subculture depending on growing state; Screening and culturing program is entered after repeatedly subculture.When noting each subculture, outwell the medium that half is old, supplement half fresh culture, be beneficial to form " conditioned medium ", promote that suspension medium embryoid increases fast.

C. meeting suspends cultivates the screening of embryoid: after 3 ~ 4 subcultures, enter screening sequence; Carefully choose close structure, in spherical, that light green color, propagation are fast embryoid, eliminate that growth is loose, the irregular bulb of form.Constantly selected by several weeks, make the culture in each 500 milliliters of triangular flasks, 200 milliliters of liquid measures, morphologically unanimously, similar on color and luster, in structure closely, the cultivating system that culture fluid is limpid.

D. than biological growth speed

Computing formula: (harvest yield one inoculum concentration) ÷ cultivates cultivated days=growth rate when weight ÷ gathers in the crops

In implementation process after testing, its growth rate reaches 8-9.5 milligram/gram sky in the present invention.

Embodiment 2. embryoid sterile dry production (see photo)

For making polysaccharide content of dendrobium candidum reach more than 10%, embryoid finished product harvest cycle is 7 days.Results embryoid finished product, after the medium that ultra-clean water rinses out appearance, room temperature dry to fresh weight moisture about 5%, put into 55 DEG C of baking ovens, be dried to absolute dry weight, after the super pulverizing of low temperature, irradiation sterilization, embryoid sterile dry.

The polysaccharide determination of embodiment 3. dendrobium nobile embryoid

1. experimental technique

A. preparation of reagents

(1) ethanolic solution, adds absolute ethyl alcohol 80ml in 20ml water.

(2) phenol solution: take purifying phenol 5.0 grams, be added to the water and dissolve and be diluted to 100ml, mixing, (solution puts 4 DEG C of refrigerators can preserve one month) for subsequent use.

(3) concentrated sulfuric acid is for subsequent use.

(4) Glucose standards storing solution: precision takes the dextrose standard sample 1.00809 being dried to constant weight at 105 DEG C, is dissolved in water and is settled to 100ml, mixing, puts 4 DEG C of Refrigerator stores.The every ml of this solution is containing 10.080mg glucose.

(5) Glucose standards uses solution: draw Glucose standards storing solution 2.00ml, be placed in 100ml volumetric flask, add water to scale, mixing, is placed in 4 DEG C of refrigerators and preserves.

B. experimental technique

(1) glucose standard curve preparation

Accurate Glucose standards of drawing uses liquid 0,0.2,0.4,0.6,0.8,1.0, (be equivalent to glucose 0,0.04032,0.08064,0.12096,0.16128,0.2016mg) be placed in the accurate supplementing water of 25ml colorimetric cylinder respectively to 2.0ml, add phenol solution 2.0ml, rotation vortex mixer mixes, carefully add concentrated sulfuric acid 10ml, carefully mix on rotation vortex mixer, put in boiling water bath and boil 15min, cooling rear spectrophotometer, to sentence blank reagent solution at 490nm wavelength be reference, and 1cm cuvette measures absorbance.

Take glucose quality as abscissa, absorbance is ordinate, paints calibration curve.Correlation coefficient is obtained by calibration curve regression curve equation figure.

Sample determination

Sample extraction

Dendrobium nobile embryoid is dried in 60 DEG C of baking ovens, pulverizes, takes about 0.1g for subsequent use.Dendrobe powder, after 95% alcohol extract, heats 90 DEG C by 20 times of volume water, 1 hour.Filter while hot, collect filtrate, reduced pressure concentration, add 4 times of volume absolute ethyl alcohol mixings, 4000rpm, 15min are centrifugal, and precipitation washes 2 times with 80% ethanol, and final sediment is dissolved in 50ml water and is stem of noble dendrobium Thick many candies solution.

Polysaccharide determination

Precision is got polysaccharide solution 2.0ml and is placed in 25ml colorimetric cylinder, add phenol solution 2.0ml, on rotation vortex mixer after mixing, mix after carefully adding concentrated sulfuric acid 10.0ml, be placed in water-bath and boil 10min, be cooled to room temperature spectrophotometer at 490nm wavelength place, take reagent blank as reference, 1cm cuvette measures Glucose standards solution and sample (by doubling dilution to suitable concn) absorbance, is glucose standard curve figure, Thick many candies content in calculation sample.

Computational methods

OD value per sample finds corresponding glucose content from calibration curve, is multiplied by extension rate, is converted into total starches content, then divided by drying the dry weight of sample, be the content containing Thick many candies in every gram of dry weight sample, unit is mg/g.

Polyoses content=OD value × extension rate × population of samples amasss/sample dry weight

2. Dendrobium officinale polysaccharide measurement result

To test and quantitative assay has been carried out to the polyoses content in the dendrobium nobile embryoid of different cultivated days and cell mass and culture supernatant thereof. result shows: institute's polyoses content in expecting of measuring and monitoring the growth of standing timber is stablized, and wherein the embryoid polyoses content of 10 to 18 days is 160-173mg/g dry weight: the 16-17.3% accounting for total amount.

Accompanying drawing explanation

Accompanying drawing, suspends and cultivates dendrobium nobile embryoid production technological process

Claims

1. an In vitro Suspension cultivates amplification stem of noble dendrobium embryoid technique, its incubation is: stem of noble dendrobium band dormancy leaf stem section, be inserted on desired solid medium, this medium adds proper amount of carbon source, appropriate Juice, appropriate agar powder, and appropriate plant growth regulator, under suitable cultivation temperature and intensity of illumination, after embryoid to be initiated grows, embryoid is peeled off, be placed on identical above-mentioned medium and increase, being transferred to above-mentioned medium after amplification removes in the liquid nutrient medium of agar powder, carry out suspension to cultivate, every bottle by suitable rate of vaccination inoculation, according to growing state subculture, after several subculture, finally reach every 7 days results once, polyoses content reaches more than 10%, than biological growth rate 8-9.5 milligram/gram sky, and the amplification system keeping the anniversary stable.

2. the embryoid of the stem of noble dendrobium described in claim 1 culture technology, it is characterized in that stem of noble dendrobium embryoid be under solid culture condition subculture screening, carry out under floating condition cultivate amplification.

3. the embryoid of the stem of noble dendrobium described in claim 1 appropriate media, as 1/4MS medium, 1/2MS medium, MS medium, 1/4MS mineral salts culture medium, 1/2MS mineral salts culture medium, MS mineral salts culture medium, White medium, B5 medium, N6 medium, SH medium, Miller medium etc.

4. add proper amount of carbon source in medium described in claim 1, comprise fructose, sucrose, maltose, mannose, glucose, comprise wherein one or more mixture.

5. add suitable fruit and vegetable juice in medium described in claim 1, comprise 2%-5% apple juice, 1%-10% bananas juice, 2%-5% tomato juice, 1%-8% potato juice, or the mixture etc. of wherein several fruit and vegetable juice.

6. the embryoid of the stem of noble dendrobium described in claim 1, is characterized in that physiologically active is 2000lx, light application time 10-20h/ days in intensity of illumination.

7. the embryoid of the stem of noble dendrobium described in claim 1 suspends and cultivates rate of vaccination is 1%-10% (W/W).

8. the embryoid of the stem of noble dendrobium described in claim 1, its polyoses content reaches more than 10%.

9. the cultivating system of the embryoid of the stem of noble dendrobium described in claim 1, is characterized in that can do modularization copies amplification, realizes suitability for industrialized production.

10. with stem of noble dendrobium embryoid dry product and the powder of explained hereafter described in claim 1, for Chinese herbal medicine formula, food additives, the preparation of functional beverage and health food.