CN107333651B - A method of improving Dendrobidium huoshanness protocorm alkaloid - Google Patents
A method of improving Dendrobidium huoshanness protocorm alkaloid Download PDFInfo
- Publication number
- CN107333651B CN107333651B CN201710533473.1A CN201710533473A CN107333651B CN 107333651 B CN107333651 B CN 107333651B CN 201710533473 A CN201710533473 A CN 201710533473A CN 107333651 B CN107333651 B CN 107333651B
- Authority
- CN
- China
- Prior art keywords
- protocorm
- alkaloid
- dendrobidium huoshanness
- elicitor
- phomopsis
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- 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
- A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
-
- 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
- A01H4/001—Culture apparatus for tissue culture
Abstract
The present invention is to provide a kind of methods for improving Dendrobidium huoshanness protocorm alkaloid, and using Dendrobidium huoshanness protocorm as material, the alkaloid of Dendrobidium huoshanness protocorm is improved using Phomopsis elicitor.The present invention has studied the influence of alkaloid when Phomopsis elicitor cultivates the suspension of Dendrobidium huoshanness protocorm, it is intended that the medicinal ingredients such as large-scale industrialized production alkaloid of Dendrobidium huoshanness provide new approaches, provide fundamental basis for the medicine source exploitation of later Dendrobidium huoshanness.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to a kind of side for improving Dendrobidium huoshanness protocorm alkaloid
Method.
Background technique
Dendrobidium huoshanness (Dendrobium huoshanenseC. Z. Tang et S. J. Cheng), it is commonly called as a meter dry measure used in former times,
For the perennial herbaceous plant that grows nonparasitically upon another plant of orchid family Dendrobium, Huoshan County, Anhui Province of Da Bie Mountain area is originated in, with being not only peace canaliculus medicine
One of material, or one of nine kinds of medicinal dendrobiums on the regular payroll are recorded by 2005 editions Pharmacopoeias of the People's Republic of China.With benefit of promoting the production of body fluid
Stomach, anti-cancer and cancer-preventing, the multiple efficacies such as relieves fatigue at the clear larynx of improving eyesight.Dendrobidium huoshanness is often grown nonparasitically upon another plant with herbaceous plant such as moss, stone reeds
Together, it is grown on the place of the cool high humidity of temperature more, therefore its growth region is extremely narrow, along with its natural breed ability is low, growth
Speed is slow, and medical value height makes the mankind excessively excavate utilization, and wild Dendrobidium huoshanness resource has remained little.Fortunately from 20th century
The end of the fifties, domestic scholar begin to the sustainable development to Dendrobidium huoshanness resource with using being investigated and ground
Study carefully.Currently used technology is rapid propagation in vitro, but since the Dendrobidium huoshanness breeding cycle is long, especially pharmaceutical ingredient content is low
Phenomenon is but unable to get always solution, reduces its medical value.
Dendrobidium huoshanness main component is alkaloid and polysaccharide, and pharmacological action and the two have close association.It is especially raw
There is voiceless sound improving eyesight, analgesic to bring down a fever, anti-cataract, protect a variety of effects such as gastrointestinal tract for alkaloids substance, be most important in dendrobium nobile
One of effective active composition.Alkaloid is a kind of alkaline organic compound, and has significant physiological activity, since it is being planted
Object is intracorporal to be widely present, thus is otherwise known as " vegetable soda ".The separation for the first time of Dendrobium Sw alkaloid is 1932 Nian You
This Suzuki show is done to be completed on HERBA DENDROBII, and it is named as dendrobine (Dendrobine).Descendant is again constantly from stone
It is extracted in dry measure used in former times platymiscium and has isolated more than ten kinds of alkaloids, 1964, dog Fu Kangfu confirmed that Dendrobium Sw Dendrobidium huoshanness is raw
The structure of alkaloids is sequiterpene skeleton structure.But content of the alkaloid in Dendrobium Sw is generally all less, and different
Its alkaloid of the dendrobium nobile of type would also vary from.Zhu Yan etc. grinds the dendrobium candidum of 17 germplasm, 41 samples
Study carefully, find its alkaloid can due to the difference of the germplasm of dendrobium nobile and Planting Years difference.Ding Yaping is the micro- Huoshan of peace
The total alkaloid content of the stem of 3 kinds of dendrobium nobiles in county is determined, as a result: Dendrobidium huoshanness 0.0291%, dendrobium candidum
0.024%, Dendrobium Moniliforme 0.028%.
Fungal elicitor is a kind of special biochemical signals, from fungi, is had single-minded, efficient, selectively
Induce the effect of the expression of certain specific genes.It, can be with extracellular special receptor albumen during the interaction with plant
Combine, then stimulate cell secretion signal molecule, and then regulate and control secondary metabolism approach related gene, thus promote synthesis,
Specific secondary metabolite in accumulator plant body.Prime report fungal elicitor is in nineteen sixty-eight, Cruickshand et al.
It will be extracted from one of the mycelium of clump stalk spore bacterium polypeptide, act on Kidney bean, it is found that this polypeptide can lure
The endocarp for leading Kidney bean forms and accumulates phaseolin, and subsequent people have carried out more extensive research to fungal elicitor.Grandson
U.S. tinkling of pieces of jade discovery fungal elicitor can promote the generation of betulin in white birch suspension cell.Wang Hong etc. is to the root of hair of sweet wormwood with 3
Fungal elicitor is planted to be handled, as a result, it has been found that can promote the accumulation of secondary metabolites qinghaosu in Hairy Root Cultures of Artemisia annua.And this
Phomopsis elicitor used by inventing be by separating Phomopsis mycelium, by cell inactivation it is broken be prepared contain
There is the sterile solution of Phomopsis mycelium and its metabolin.Phomopsis elicitor is as one of those kind of fungal elicitor
Class, can promote plant to generate metabolite, this and Xu little Ping can significantly improve longan embryo about P. flougzuoe elicitor
The result of study of polyoses content in property callus is consistent.
It is that raising plant tissue is thin it can be seen that adjusting the secondary metabolism approach of plant by addition fungal elicitor
The important way of secondary metabolite yield in born of the same parents' culture.The present invention is lured using the mycelia extract of Phomopsis as fungi
Guide inquires into the different disposal of Dendrobidium huoshanness come the influence of the content to Dendrobidium huoshanness alkaloid is further by it,
To lay the foundation for the large-scale industrialized production secondary metabolites of Dendrobidium huoshanness.
The suspension culture of plant cell be developed from the Liquid Culture of plant callus basis it is a kind of higher
The training method of effect makes full use of Dendrobidium huoshanness protocorm easily to disperse, proliferative speed is fast, is metabolized the characteristic of totipotency to carry out liquid
The body culture that suspends overcomes the limitation that rooting of vitro seedling is weak, transplanting is difficult, can increase substantially Protocorm Multiplication rate and its
Pharmaceutical ingredient content is the big important channel for solving the medicinal resource scarcity of plant.
Summary of the invention
The purpose of the present invention is to provide a kind of methods for improving Dendrobidium huoshanness protocorm alkaloid.
To achieve the above object, the present invention adopts the following technical scheme:
A method of Dendrobidium huoshanness protocorm alkaloid is improved, Dendrobidium huoshanness protocorm is put in containing quasi- stem point
It is cultivated in the culture medium of mould elicitor.
Specific method includes the following:
(1) the Dendrobidium huoshanness protocorm of long-term preservation is taken, taking-up, which is placed in 1/2 solid medium, cultivates subculture, screens
It grows fine out, the protocorm of no differentiation；
(2) the Dendrobidium huoshanness protocorm filtered out is inoculated in 1/2 MS fluid nutrient medium, is 2000 lx in intensity of illumination
Under conditions of, liquid shake culture is carried out to Dendrobidium huoshanness protocorm with the revolving speed of 110 r/min with shaking table, medium pH is
5.8, the cultivation cycle of Dendrobidium huoshanness bulb is 45 d, primary every 45 d subcultures, prevents protocorm from occurring breaking up or browning；
(3) non-activated Phomopsis strain inoculated is subjected to activation rejuvenation culture 1 week in PDA culture medium；It selects quasi-
The bacterium colony that Phoma sp grows fine is transferred in the above-mentioned PDA liquid medium for not adding agar, in dark, 28 DEG C, 110 r/
Under the environmental condition of min, a large amount of Multiplying cultures are carried out 2 weeks, obtain Phomopsis elicitor；
(4) the Dendrobidium huoshanness protocorm for obtaining step (2) is in 1/2 MS+NAA of culture medium, 0.1 mg/L+KT 0.05
Cultivated in mg/L+20 g/L sucrose, pH is adjusted to 5.4, while Phomopsis elicitor 0-300 mg/L is added, in temperature be 25 ±
2 DEG C, light application time is 12 h/d, and intensity of illumination is about 2000 lx, and shaking speed cultivates 5-11 under conditions of being 110 r/min
d。
The present invention has the advantages that handling 7 d of Dendrobidium huoshanness protocorm using 250 mg/L Phomopsis elicitor concentration
When its alkaloid can reach 0.09033%.Therefore, using Phomopsis induction subprocessing Dendrobidium huoshanness protocorm to its work
Factoryization large-scale production alkaloid is of great significance.
Detailed description of the invention
Fig. 1 Phomopsis rejuvenation culture 1 week.
Fig. 2 Phomopsis Multiplying culture 2 weeks.
Specific embodiment
1 materials and methods
1.1 test material
The material of this test is external evoked and long-term preservation Dendrobidium huoshanness protocorm, derives from University Of Agriculture and Forestry In Fujian garden
Skill plant biological engineering research institute.Taking-up is placed in 1/2 solid medium and cultivates subculture, filters out and grows fine, no differentiation
Protocorm as test material.
1.2 test method
1.2.1 Liquid Culture
The Dendrobidium huoshanness protocorm that early period cultivates and filters out on 1/2 MS solid medium is inoculated in 1/2 MS liquid
In body culture medium, under conditions of intensity of illumination is about 2000 lx, with shaking table with the revolving speed of 110 r/min to Dendrobidium huoshanness original
Bulb carries out liquid shake culture, and medium pH is adjusted to 5.8 before high pressure, and culture vessel uses 200 mL conical flasks, fills liquid
Amount is 60 mL or so.The cultivation cycle of Dendrobidium huoshanness elicitor is 45 d, therefore should be primary every 45 d subcultures, prevents protocorm
Stem occurs breaking up or browning.
1.2.2 the preparation of fungi Phomopsis elicitor
By the strain inoculated of non-activated Phomopsis in PDA culture medium (200 g of potato, 20 g of glucose, agar 20
g、H21 L of O) in carry out activation rejuvenation culture 1 week, such as Fig. 1；Selecting the bacterium plate that Phomopsis grows fine, to be divided into size suitable
In fritter be transferred in the above-mentioned PDA liquid medium for not adding agar respectively, in dark, 28 DEG C, the ring of 110 r/min
Under the conditions of border, a large amount of Multiplying cultures are carried out 2 weeks, such as Fig. 2；Decompression suction filtration is carried out to the culture medium of acquisition and removes bacteria-removing liquid, and is used
Distilled water repeats to filter 3 times, weighs 5 g mycelia cell homogeniser crushing grindings, and be settled to 100 mL in 121 DEG C of high pressures
Under the conditions of sterilize after 20 min, be placed in stored for future use in 4 DEG C of refrigerators.Total sugar content in gained bacterium solution is measured with Phenol sulfuric acid procedure
Concentration, and demarcate with this concentration the concentration of bacterium solution.
1.2.3 the processing method of sample
This test by force optimizes Dendrobidium huoshanness protocorm in vitro culture using Dendrobidium huoshanness protocorm as material, according to Lv Kai
Optimum condition: 1/2 MS+NAA, 0.1 mg/L+KT, 0.05 mg/L is minimal medium, 20 g/L sucrose is added, pH is adjusted to
5.4, and needed by test process, Phomopsis elicitor is added, concentration is respectively 0,100,150,200,250,300 mg/L,
Access the Dendrobidium huoshanness protocorm of 4.0 g in the 200 mL conical flasks containing 50 mL processing culture medium, in temperature be 25 ± 2
DEG C, light application time is 12 h/d, and intensity of illumination is about 2000 lx, shaking speed cultivates 5 respectively under conditions of being 110 r/min,
7,9,11 d.It, will be former after resulting Dendrobidium huoshanness protocorm is cleaned 3 ~ 5 times and cleaned with distilled water after test material harvest
Bulb is placed in thermostatic drying chamber, 60 DEG C at a temperature of carry out being dried to weight and remain unchanged, then by the examination after drying
It tests material to be crushed with micromill, crosses 40 meshes, be sealed in the hermetic bag for being placed with desiccant, for use.
1.2.4 the production of alkaloid standard curve
Using Wan withAcid-dye Colorimetry alkaloid.Precision weighs 1.00 mg dendrobine reference substances, molten with chloroform
It is settled to 100 mL after solution, draws 1.0,2.0,3.0,4.0,5.0 mL dendrobium nobile aqueous slkalis respectively and is added to 60 mL separatory funnels
In, supplement chloroform to volume is 10.0 mL.5.0 mL neck 4.6,1.0 mL bromine cresols of phthalic acid potassium-sodium hydroxide PH is added
Green 0.04 % solution acutely shakes 3 min, stands 30 min.Chloroform layer is filtered with chloroform and the absorbent cotton of drying, takes subsequent filtrate
6.0 mL are added 0.01 mol/L sodium hydroxide ethanol solution, 1.0 mL, stand after shaking up.To be not added with dendrobine control
Product solution be blank control, with ultraviolet specrophotometer under 620 nm wavelength colorimetric estimation light absorption value.With the dendrobium nobile after dilution
Alkali concentration is abscissa, and the absorbance of sample is that ordinate makes standard curve.
1.2.5 the measurement of Dendrobidium huoshanness protocorm alkaloid
Precision weighs 0.4 g of Dendrobidium huoshanness protocorm dried powder sample and is put into 60 ml ground conical flasks, with appropriate
The close plug of (2.0 ml) concentrated ammonia liquor infiltrates 30 min.10.0 ml of chloroform, weighing record is added.60 DEG C of ultrasonic oscillations extract 30
Min is supplemented chloroform to original weight.Filtering takes 2.0 ml of subsequent filtrate, and subsequent filtrate is added in 60 ml separatory funnels, adds chloroform
8.0 mL.0.04% solution of bromocresol green of the Potassium Hydrogen Phthalate pH4.6 and 1.0 mL of 5.0 mL is added, by separatory funnel
Acutely 3 min of concussion stand 30 min.Chloroform layer is filtered, 6.0 mL of subsequent filtrate is taken, the 0.01 mol/L hydrogen-oxygen of 1.0 mL is added
Change sodium ethanol solution, fullys shake and stood after shaking up.Using be not added with dendrobine reference substance solution as blank control,
With ultraviolet specrophotometer under 620 nm wavelength colorimetric estimation light absorption value.
Referring to above-mentioned standard curve, by determine come light absorption value, calculate under corresponding light absorption value in standard solution
Dendrobidium huoshanness alkaloid, with formula: alkaloid (%)=C * D/W calculates extracted Dendrobidium huoshanness biology
Alkali content, wherein C be sample solution in dendrobine concentration, D be sample solution extension rate, W for institute's sample reality
Weight.
1.2.6 data are analyzed
This test takes SPSS 19.0 and EXCEL data processing software to be made a concrete analysis of to obtained data, always
Knot.
2 results and analysis
2.1 standard curve Regression Equations
The light absorption value measured under 620 nm wavelength by ultraviolet specrophotometer, the alkaloid standard thus drawn are bent
Line, it is y=0.0046x+0.005 that statistical analysis, which obtains standard curve regression equation, and R2=0.9975 illustrates made
It can be used for the measurement of Dendrobidium huoshanness alkaloid as standard curve.
2.2 Phomopsis elicitor various concentrations handle the analysis that 5 d influence protocorm alkaloid
Found by statistical analysis, Dendrobidium huoshanness protocorm handles to 5 d under Phomopsis elicitor various concentration and
After measure its alkaloid, the results are shown in Table 1；Through table 1 it is found that when Phomopsis elicitor concentration is 250 mg/L,
Alkaloid highest is 0.051%；When Phomopsis elicitor concentration is 0 mg/L, alkaloid is minimum, is
0.00633%.In the case where handling number of days is 5 d, the content of Dendrobidium huoshanness protocorm alkaloid is dense with Phomopsis elicitor
The raising of degree shows downward trend after first rising.
The content of protocorm alkaloid when table 1 Phomopsis elicitor, 5 d of different dense processing
2.3 Phomopsis elicitor various concentrations handle the analysis that 7 d influence protocorm alkaloid
Found by statistical analysis, Dendrobidium huoshanness protocorm handles to 7 d under Phomopsis elicitor various concentration and
After measure its alkaloid, the results are shown in Table 2 Hes；Through table 2 it is found that when Phomopsis elicitor concentration is 250 mg/L,
Its alkaloid highest is 0.09033%；When Phomopsis elicitor concentration is 0 mg/L, alkaloid is minimum,
It is 0.013%.In the case where handling number of days is 7 d, the content of Dendrobidium huoshanness protocorm alkaloid is dense with Phomopsis elicitor
The raising of degree shows two peak values, but overall downward trend after showing first rising.
The content of protocorm alkaloid when 2 Phomopsis elicitor various concentration of table handles 7 d
2.4 Phomopsis elicitor various concentrations handle the analysis that 9 d influence protocorm alkaloid
Found by statistical analysis, Dendrobidium huoshanness protocorm handles to 9 d under Phomopsis elicitor various concentration and
After measure its alkaloid, the results are shown in Table 3；Through table 3 it is found that when Phomopsis elicitor concentration is 200 mg/L,
Alkaloid highest is 0.07%；When Phomopsis elicitor concentration is 0 mg/L, alkaloid is minimum, is
0.017%.In the case where handling number of days is 9 d, the content of Dendrobidium huoshanness protocorm alkaloid is with Phomopsis elicitor concentration
Raising show two peak values, but it is overall show first rise after downward trend.
The content of protocorm alkaloid when 3 Phomopsis elicitor various concentration of table handles 9 d
2.5 Phomopsis elicitor various concentrations handle the analysis that 11 d influence protocorm alkaloid
Found by statistical analysis, Dendrobidium huoshanness protocorm handles to 11 d under Phomopsis elicitor various concentration and
After measure its alkaloid, the results are shown in Table 4；Through table 4 it is found that when Phomopsis elicitor concentration is 200 mg/L,
Alkaloid highest is 0.0903%；When Phomopsis elicitor concentration is 0 mg/L, alkaloid is minimum, is
0.01233%.In the case where handling number of days is 11 d, the content of Dendrobidium huoshanness protocorm alkaloid is with Phomopsis elicitor
The raising of concentration shows downward trend after first rising.
The content of protocorm alkaloid when 4 Phomopsis elicitor various concentration of table handles 11 d
The totality point that 2.6 Phomopsis elicitor various concentrations and different disposal number of days influence protocorm alkaloid
Analysis
Found by test data analyzer, by Dendrobidium huoshanness protocorm be inoculated in respectively Phomopsis elicitor concentration be 0,
100, in the liquid suspension culture base of 150,200,250,300 mg/L, its alkaloid is counted after cultivating 5,7,9,11 d,
It the results are shown in Table 5.It can be seen from Table 5 that Phomopsis elicitor concentration is 250 mg/L, processing number of days is protocorm under 7 d
Alkaloid highest is 0.09033%；The processing group of Phomopsis elicitor culture is added than being not added with elicitor culture
Alkaloid it is high, illustrating Phomopsis elicitor to a certain extent can promote Dendrobidium huoshanness protocorm alkaloid to contain
The generation and accumulation of amount.Under same elicitor concentration, after first increase is presented with the increase of processing number of days in protocorm alkaloid
Downward trend；Under same processing number of days, after first rising is totally presented with the increase of elicitor concentration in protocorm alkaloid
Downward trend.
The content of protocorm alkaloid under 5 Phomopsis elicitor various concentration of table and different disposal number of days
Main effect variance analysis (being shown in Table 6) is carried out to test result, as a result, it has been found that processing number of days, concentration for the treatment of and the two
Interactive conspicuousness P value be 0, less than 0.01, illustrate fungal elicitor processing number of days, concentration and the two interaction
It acts on the influence to Dendrobidium huoshanness protocorm alkaloid and all has extremely significant sex differernce in 0.01 level.By comparing
The F of these factors is worth size it is found that the primary and secondary sequence for influencing Dendrobidium huoshanness protocorm alkaloid accumulation factor is to lure
Guide concentration > processing number of days > processing number of days * concentration.In summary, the accumulation of Dendrobidium huoshanness protocorm alkaloid is influenced most
Big factor is elicitor concentration, and the smallest is processing day Particle density.
The main effect that the different disposal number of days and concentration of 6 Phomopsis elicitor of table influence protocorm alkaloid
It examines
To 24 processing be described with Multiple range test analyze, then filter out optimal processing and with its there is no conspicuousness it is poor
Different processing is described and Multiple range test, is finally selected most beneficial for the best of Dendrobidium huoshanness protocorm alkaloid accumulation
Processing.
Discovery (table 6) is examined through F, there are reciprocations between three factors in test, therefore select most in 24 processing
The optimization process for being conducive to the accumulation of Dendrobidium huoshanness protocorm alkaloid, by the description of each processing of the following table 3 it is found that optimal
Processing is processing 11, and alkaloid mean value is 0.09033%.
The description that the different disposal number of days and concentration of 7 Phomopsis elicitor of table influence protocorm alkaloid
Homogeneity variance is carried out with 19.0 software of SPSS to the experimental result of acquisition and examines (see Table 8), can analyze
Know: the Phomopsis elicitor of different disposal number of days and various concentration has the accumulation of Dendrobidium huoshanness protocorm alkaloid
Significant impact.Since conspicuousness P value is 0.010, less than 0.05, illustrate that there are significant differences between each processing, there is no sides
Difference is homogeneity.
The variance that the different disposal number of days of 8 Phomopsis elicitor of table and concentration influence protocorm alkaloid is neat
Property examine
Since variance is there is no homogeneity, compared two-by-two with Dunnett T3 method.It can by analysis before
Optimization process is known to handle 11, therefore processing 11 and Multiple range test tables of other processing is selected to be analyzed, and passes through the following table 9, table
10 it is found that processing 11 and processing 10, processing 12, processing 16, processing 22, handle 23 conspicuousness P value be respectively 0.064,
0.991,0.274,0.919,0.091. is all larger than 0.05, illustrates processing 11 and processing 10,12,16,22,23 pairs of Huoshan stones of processing
Significant difference is not present in the accumulation of dry measure used in former times protocorm alkaloid；Processing 11 and processing 5, processing 18, the conspicuousness P for handling 21
Value is respectively 0.012,0.030,0.016, is greater than 0.01, less than 0.05, illustrates 5,18,21 pairs of Dendrobidium huoshanness of processing 11 and processing
There are significant differences for the accumulation of protocorm alkaloid, but extremely significant sex differernce is not present；And handle 11 and 24 processing
In other processing P values be respectively less than 0.01, illustrate processing 11 with other processing to Dendrobidium huoshanness protocorm alkaloid
Accumulation there are extremely significant sex differernces.Therefore, processing 5,10,11,12,16,18,21,22 and 23 is most beneficial for Dendrobidium huoshanness
The processing of protocorm alkaloid accumulation.
The multiple ratio that the different disposal number of days and concentration of 9 Phomopsis elicitor of table influence protocorm alkaloid
Compared with
* the significance of equal value difference is 0.05.
The different disposal number of days and concentration of 10 Phomopsis elicitor of table influence protocorm alkaloid multiple
Compare
* the significance of equal value difference is 0.01.
By analyze above it is found that optimal processing and with its there is no significant difference processing for processing 5,10,11,
12,16,18,21,22 and 23, this 9 processing concrete condition such as the following table 11, comprehensively consider large-scale industrialized production when
Between, cost problem, then when Phomopsis elicitor concentration be 250 mg/L culture medium in handle 7 d be most beneficial for Huoshan
The processing of dendrobium nobile protocorm alkaloid accumulation.
11 optimal processing situation of table screens table
3 discuss
The accumulation of 3.1 Phomopsis elicitors promotion Dendrobidium huoshanness protocorm alkaloid
This experimental study discovery is added to the blank group of the processing group of Phomopsis elicitor than being not added with elicitor
Alkaloid is high；It is raw that 250 mg/L Phomopsis induction, 7 d of Dendrobidium huoshanness protocorm can significantly improve Dendrobidium huoshanness protocorm
Alkaloids content can reach 0.09033%.The measured highest alkaloid (0.09033%) of this test is above Ding Yaping
(0.0291%), (0.0261% alkaloid measured, elicitor is as in Plant Tissue Breeding in Dendrobidium huoshanness by Luo Jianping
A kind of additive can effectively adjust the secondary metabolism approach of plant, fungal elicitor by the enzymatic activity of adjusting plant cell
The production of secondary metabolites in plant callus or suspension cell is had been widely used at present.This Preliminary Experiment has inquired into quasi- stem
The influence of alkaloid when the mould elicitor of point cultivates the suspension of Dendrobidium huoshanness protocorm, it is intended to be Dendrobidium huoshanness large-scale plant
Metaplasia generates the medicinal ingredients such as alkaloids and provides new approaches, provides fundamental basis for the medicine source exploitation of later Dendrobidium huoshanness.
This test result shows that Phomopsis elicitor can significantly improve the content of Dendrobidium huoshanness protocorm alkaloid, may
The reason is that: alkaloid is metabolite relevant to resistance in plant, and contains active material in Phomopsis elicitor, these
Certain ingredients in active material may be intracellular induction of Dendrobidium huoshanness protocorm defense response, accelerate its secondary metabolism way
Diameter promotes secondary metabolites, for example the accumulation of alkaloid, and peptide is added in parsley and lures for the discovery such as this and Hahlbrock
Its gene related with defense response of guide is activated and may have similar effect.
The Phomopsis of 3.2 various concentrations and number of days induction subprocessing Dendrobidium huoshanness protocorm will affect alkaloid
Accumulation
Test result finds that the Phomopsis induction subprocessing of various concentration and different number of days is raw to Dendrobidium huoshanness protocorm
Alkaloids content has opposite impacts on.Under identical cultivated days, Dendrobidium huoshanness protocorm alkaloid is dense with elicitor
Downward trend after first rising is totally presented in the increase of degree, this is consistent with the conclusion that Lin Yanjun is obtained.Low concentration Phomopsis
Elicitor (100 mg/L, 150 mg/L) is not very significant to the facilitation of Dendrobidium huoshanness protocorm alkaloid, Ke Nengyuan
Because being the too low process for influencing intercellular signal conduction of elicitor concentration.In the case where Phomopsis elicitor is with concentration conditions, with
Downward trend after first rising is totally presented in the increase of cultivated days, the content of Dendrobidium huoshanness protocorm alkaloid.In protocorm
Early growth period, it is also necessary to new media environment is gradually adapted to, it is at this moment more blunt to the stimulate the reaction of Phomopsis elicitor,
Metabolism is also relatively slower；After protocorm adaptation, Phomopsis elicitor can participate in secondary metabolism by changing protocorm
The activity of the enzyme of approach improves the content of its alkaloid；And when in the case where inducing subprocessing, incubation time too long, intends stem to protocorm
The mould elicitor of point may generate toxic action to the cell of protocorm, to influence the synthesis and accumulation of its alkaloid.
Fungal elicitor induces the synthesis of plant secondary metabolites mainly to be induced subcategory, concentration, induction time by it
And addition period etc. influences.This experimental study Phomopsis elicitor various concentration and different induction times are to Huoshan stone
The influence of dry measure used in former times protocorm alkaloid, but be not directed to elicitor and influence of the period to its alkaloid is added.Brodelius
It indicates, fungal elicitor is more significant in the cell growth phase effect of Plant Tissue Breeding.To be further Dendrobidium huoshanness work
Factoryization is proliferated on a large scale and the production of secondary metabolites contributes, and can study the different growing stage in Dendrobidium huoshanness protocorm
Addition fungal elicitor grows it and what the accumulation of secondary metabolites has influence.
The foregoing is merely presently preferred embodiments of the present invention, all equivalent changes done according to scope of the present invention patent with
Modification, is all covered by the present invention.
Claims (2)
1. a kind of method for improving Dendrobidium huoshanness protocorm alkaloid, it is characterised in that: Dendrobidium huoshanness protocorm to be put in
It is cultivated in culture medium containing Phomopsis elicitor.
2. a kind of method for improving Dendrobidium huoshanness protocorm alkaloid according to claim 1, it is characterised in that: tool
Body method includes the following:
(1) the Dendrobidium huoshanness protocorm of long-term preservation is taken, taking-up, which is placed in 1/2 solid medium, cultivates subculture, filters out length
Gesture is good, the protocorm of no differentiation；
(2) the Dendrobidium huoshanness protocorm filtered out is inoculated in 1/2 MS fluid nutrient medium, in the item that intensity of illumination is 2000 lx
Under part, liquid shake culture is carried out to Dendrobidium huoshanness protocorm with the revolving speed of 110 r/min with shaking table, medium pH 5.8, suddenly
The cultivation cycle of mountain dendrobium nobile bulb is 45 d, primary every 45 d subcultures, prevents protocorm from occurring breaking up or browning；
(3) non-activated Phomopsis strain inoculated is subjected to activation rejuvenation culture 1 week in PDA culture medium；Select quasi- stem point
The mould bacterium colony to grow fine is transferred in the above-mentioned PDA liquid medium for not adding agar, in dark, 28 DEG C, 110 r/min
Environmental condition under, carry out a large amount of Multiplying cultures 2 weeks, obtain Phomopsis elicitor；
(4) the Dendrobidium huoshanness protocorm for obtaining step (2) is in 1/2 MS+NAA of culture medium, 0.1 mg/L+KT, 0.05 mg/L+
Cultivated in 20 g/L sucrose, pH is adjusted to 5.4, while Phomopsis elicitor 100-300 mg/L is added, in temperature be 25 ± 2
DEG C, light application time is 12 h/d, and intensity of illumination is 2000 lx, and shaking speed cultivates 5-11 d under conditions of being 110 r/min.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710533473.1A CN107333651B (en) | 2017-07-03 | 2017-07-03 | A method of improving Dendrobidium huoshanness protocorm alkaloid |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710533473.1A CN107333651B (en) | 2017-07-03 | 2017-07-03 | A method of improving Dendrobidium huoshanness protocorm alkaloid |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107333651A CN107333651A (en) | 2017-11-10 |
| CN107333651B true CN107333651B (en) | 2019-05-10 |
Family
ID=60218963
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710533473.1A Active CN107333651B (en) | 2017-07-03 | 2017-07-03 | A method of improving Dendrobidium huoshanness protocorm alkaloid |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107333651B (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111893206B (en) * | 2020-08-20 | 2021-07-27 | 华南农业大学 | Identification method and kit for dendrobium material with high dendrobine content |
| CN112005884B (en) * | 2020-09-14 | 2022-02-22 | 广西壮族自治区药用植物园 | Method for improving alkaloid content of subprostrate sophora tissue culture seedling by utilizing selenium |
| CN112154914A (en) * | 2020-10-09 | 2021-01-01 | 广西壮族自治区药用植物园 | Method for improving alkaloid content of subprostrate sophora tissue culture seedling by using calcium ions |
| CN113025498A (en) * | 2021-02-05 | 2021-06-25 | 遵义医科大学 | Method for co-culturing endophyte and dendrobium nobile protocorm with stable and high dendrobine yield |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101358179A (en) * | 2008-09-25 | 2009-02-04 | 东北林业大学 | Method for improving gallic acid content in amur maple cell using elicitor |
| CN101824459B (en) * | 2009-12-23 | 2015-05-20 | 东北林业大学 | Method for promoting accumulation of triterpene in betula platyphylla suk. suspension cell by utilizing endophytic fungi elicitor |
| CN102154116B (en) * | 2010-12-22 | 2013-06-12 | 浙江大化生物科技股份有限公司 | Endophytic fungus Phomopsis sp. and use thereof |
| CN104904491B (en) * | 2014-03-10 | 2017-07-28 | 廉美兰 | A kind of method that utilization ginseng pathogenic bacterium inducing improves saponin content in bioreactor culture American Ginseng adventitious root |
| CN105886452A (en) * | 2014-12-25 | 2016-08-24 | 廉美兰 | Method for improving active substances in protocorm of dendrobium officinale kimura et migo by virtue of abiotic elicitors |
| CN105906641B (en) * | 2016-05-20 | 2017-12-05 | 皖西学院 | A kind of method of suspended culture cell production Dendrobidium huoshanness alkaloid |
-
2017
- 2017-07-03 CN CN201710533473.1A patent/CN107333651B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN107333651A (en) | 2017-11-10 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107333651B (en) | A method of improving Dendrobidium huoshanness protocorm alkaloid | |
| Fulzele et al. | Production of terpenoid from Artemisia annua L. plantlet cultures in bioreactor | |
| JPH08500973A (en) | Taxus, related taxanes and yew cultures as raw materials for other novel anti-cancer / anti-viral compounds | |
| CN102835245B (en) | Bionic culture method for cordyceps sobolifera | |
| CN104920065B (en) | A kind of wild Dictyophora rubrovalvata parent species preparation method of Mount Fanjing | |
| CN108208016B (en) | Application of the volatile materials that streptomyces fimicarius generates in control of plant disease | |
| CN104604672B (en) | A kind of method of rapid screening high-nicotine tobacco mutant body | |
| Wadegaonkar et al. | Direct rhizogenesis and establishment of fast growing normal root organ culture of Withania somnifera Dunal | |
| CN109507368A (en) | A kind of accurate Resistance Identification method of seedling stage sweet potato black rot | |
| CN107629967B (en) | Liquid culture medium of germination bacteria for gastrodia elata planting and gastrodia elata planting method | |
| CN105265312B (en) | A kind of method that active principle in bioreactor culture elatus Nakai adventitious root is improved using elicitor | |
| CN102898493A (en) | Method for culturing ginseng adventitious roots to produce ginsenosides by using small simple bioreactor | |
| CN102550490A (en) | Method of raising ectropis oblique warren through sambucus chinensis and Ectropis grisescens nucleopolyhedrovirus (EcobNPV) propagation method utilizing same | |
| Kang et al. | Rapid in vitro adventitious shoot propagation of Scopolia parviflora through rhizome cultures for enhanced production of tropane alkaloids | |
| Li et al. | Production of podophyllotaxin by root culture of Podophyllum hexandrum Royle | |
| CN101358180B (en) | Method for producing triptolide and alkaloids by tripterygium wilfordii cell culture method | |
| CN103146638B (en) | Induced proliferation method for glehnia littoralis hairy roots | |
| CN102893858B (en) | Method for production of useful secondary metabolites via culturing aralia elata somatic embryos | |
| Widiyastuti et al. | Photoperiod effect on the growth and artemisinin content of Artemisia Annua grown in tropical region | |
| CN108029553A (en) | Biological engineering's method of rice dry measure used in former times | |
| CN105794632B (en) | A kind of centella polyploid and its induced breeding method | |
| Krishnan et al. | Establishment of cell suspension culture in Marchantia linearis Lehm & Lindenb. for the optimum production of flavonoids | |
| CN103361276B (en) | Saccharopolyspora spinosa HBERC-25376, culturing method thereof as well as separation method and application of active substances thereof | |
| CN107723249B (en) | Armillaria mellea liquid culture medium for planting gastrodia elata | |
| CN109906941A (en) | A kind of method of tuniclike psammosilene root adventitious root suspension culture |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |