CN101558740A - Method for hormone-free scale culture and production of dendrobium officinale embryo dry product - Google Patents
Method for hormone-free scale culture and production of dendrobium officinale embryo dry product Download PDFInfo
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- CN101558740A CN101558740A CNA2009100516635A CN200910051663A CN101558740A CN 101558740 A CN101558740 A CN 101558740A CN A2009100516635 A CNA2009100516635 A CN A2009100516635A CN 200910051663 A CN200910051663 A CN 200910051663A CN 101558740 A CN101558740 A CN 101558740A
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Abstract
The invention relates to a method for hormone-free scale culture and production of a dendrobium officinale embryo dry product, which comprises the following steps: (1) culture and inoculation of an explant; (2) subculturing multiplication of an embryoid; (3) hormone-free first-order liquid culture and screening; (4) hormone-free second-order aerated culture; (5) hormone-free ultimate 20-liter aerated culture; and (6) post treatment of a fresh finished product. The method has the advantages of simple steps, short production cycle, low cost, environmental protection and suitability for industrial production, and solves the problem of dendrobium officinale resource shortage.
Description
Technical field
The invention belongs to the preparation field of dendrobium officinale embryo dry product, particularly relate to a kind of method of hormone-free scale culture and production of dendrobium officinale embryo dry product.
Background technology
The stem of noble dendrobium always is subjected to the attention of medical circle as traditional rare traditional Chinese medicine, because its special efficacy function is subjected to physician's high praise.The stem of noble dendrobium is comparatively harsh to the production environment condition, poor growth, and biological yield is very low, add long-term uncontrolled the excavating of people, Ecological environment worsening faces whole medicinal dendrobium class plant and is on the point of the condition of going out, wherein, have another name called Dendrobidium huoshanness (Chinese medicine voluminous dictionary 1985) for excellent especially with dendrobium candidum.
For dendrobium candidum (Dendrobium officinale Kimura et Migo) plumule dry product, do not see the method that the hormone-free scale cultivation is produced up to now as yet.
Summary of the invention
Technical problem to be solved by this invention provides a kind of method of hormone-free scale culture and production of dendrobium officinale embryo dry product, and this method step is simple, and time production cycle is short; cost is low; environmentally friendly, be fit to suitability for industrialized production, solved the problem of dendrobium candidum shortage of resources.
The method of a kind of hormone-free scale culture and production of dendrobium officinale embryo dry product of the present invention comprises:
(1) cultivation of explant and inoculation
Dendrobium candidum original seed plant is cultivated into the 3-5cm height, band 3-5 sheet leaf, the adult strain of tool 3-4 internode, gather explant and wash repeatedly till the non-foam,, cut full leaf and plant base portion 0.3～0.6cm then with its sterilization with running water, be inoculated into the M2 solid culture medium, under dark condition of culture, 24 ℃ ± 1 ℃, the growth of induced dormancy bud 35～45 days;
(2) shoot proliferation of embryoid
After inducing sleeping bud successfully, move on to 2000Lx under the light training condition, illumination in 16～20 hours, peel off sleeping bud when growing to 0.8～1.2cm, be moved in the M2 liquid nutrient medium (removing the agar powder in the solid), per 7 days subcultures once, 24 ℃ ± 1 ℃, nature room light intensity after 45-50 days, induces embryoid;
(3) no hormone one-level is cultivated and screening
Getting above-mentioned embryoid is inoculated in the M2 liquid nutrient medium, carry out the level liquid enlarged culture, per 10 days subcultures once, each subculture enlarged colony by 1: 2, through 3-5 all after date, selected that the form growth is normal, green, the big or small close cultivation of profile system, use the M0 liquid nutrient medium, per 10 days subcultures once carry out 5～8 times successive transfer culture, get mature cell system;
(4) no hormone secondary aerobic culture
System is inoculated into the M0 liquid nutrient medium with above-mentioned mature cell, and adds the conditioned medium (the old medium of former female bottle) after one-level is cultivated, and carries out secondary ventilation enlarged culture, illumination in 14-16 hour is provided every day or takes sun natural daylight, cultivates 15～20 days;
(5) the ultimate aerobic culture of no hormone
Select 20 liters of culture vessels, each container liquid amount is 16 liters, wherein 14 be upgraded to fresh M0 liquid nutrient medium, the conditioned medium (the old medium of former female bottle) that 2 are upgraded to step (4) when cultivating, the seed of selecting step (4) to obtain is inoculated, keep the 2000-2500Lx luminous intensity, on autocratic culturing rack, cultivated 15～20 days, collect bright product by 38-40 degree inclined-plane;
(6) post processing of bright finished product
After above-mentioned bright product are collected, do, weigh,, press 250g, 500g, 1000g or 5000g quantitative package then in 50～60 ℃ of dryings with drench behind the purified rinse water one time, after alpha ray is sterilized, 8 ℃ of-12 ℃ of preservations.
M2 solid culture medium described in the step (1) is 1/2MS macroelement+full dose molysite+full dose trace+full dose organic principle+sucrose 30g/L+CH (caseinhydrolysate) 500mg/L+ methyl NAA2mg/L+ basic element of cell division BA0.2mg/L+100g bananas juice+agar powder 4.8g/L, PH5.8, conventional sterilization;
Sterilization described in the step (1) is after 12-15 minute → aqua sterilisa washes several times in 50% ethanol infiltration 3-5 second → 20% " 84 " liquid 7-8 minute → 0.1% mercuric chloride liquid, suck dry moisture;
M2 liquid nutrient medium described in step (2) and (3) is a large amount of element of 1/2MS+full dose molysite+full dose trace+full dose organic principle+sucrose 30g/L+CH (caseinhydrolysate) 500mg/L+ methyl NAA2mg/L+ basic element of cell division BA0.2mg/L, PH5.8, conventional sterilization;
M0 liquid nutrient medium described in the step (4) is a large amount of element of 1/2MS+full dose molysite+full dose trace+full dose organic principle+sucrose 30g/L, PH5.8, conventional sterilization;
Conditioned medium described in the step (4) is the old medium after cultivating 10～15 days in the M0 liquid nutrient medium of the stem of noble dendrobium in step (3);
Conditioned medium described in the step (5) is the old medium after cultivating 10～15 days in the M0 liquid nutrient medium of the stem of noble dendrobium in step (4);
Autocratic culturing rack described in the step (5) takes up an area of 1M for each shelf
2, adorn three layers, 3 20L containers of a row, each shelf is placed 18.
The present invention adopts the macroelement of MS and the mineral nutrition that trace element acts on culture; Sugar provides and builds up cell wall-cellulosic source as the carbon source of plant cell, simultaneously medium is formed certain osmotic pressure, so that culture starts growth; CH (caseinhydrolysate) replenishes as organic nitrogen; Bananas juice can replenishing vitamins class material, the improvement growth conditions.
Beneficial effect
Method step of the present invention is simple, and time production cycle is short, and cost is low, and is environmentally friendly, is fit to suitability for industrialized production, has solved the problem of dendrobium candidum shortage of resources.
Description of drawings
Fig. 1 is no hormone aerobic cultivation algam dendrobium dry product raw material flow sheet.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after the content of having read the present invention's instruction, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Embodiment 1
(1) preparation of explant, medium are made, inoculation:
(1) chooses long 3-5 sheet leaf, the thick 0.3-0.5 of the stem dendrobium candidum strain body of growing up that has;
(2) explant sterilization program:
After explant is gathered, be placed on the little triangular flask of 250ml, drip and go up a liquid detergent 1-2 and drip, wash repeatedly till the non-foam with running water.On super-clean bench, enter sterilizing program: after 12-15 minute → aqua sterilisa washes several times in 50% ethanol infiltration 3-5 second → 20% " 84 " liquid 7-8 minute → 0.1% mercuric chloride liquid, be put on the infiltration aseptic paper and blot attached water by following step;
(3) medium is made, is prepared:
Choosing " MS " basis, with 1/2 macroelement+iron complete+little complete+complete organic routine+sucrose 30g/L+100g bananas juice 10%/L+ agar powder 4.8g/L PH5.8, conventional sterilization, divide then and install to (sterilization in advance) in the 150ml triangular flask, (it is solid to be called for short M2 for every liter of packing 18-20 bottle, liquid just removes agar, is called for short M2);
(4) explant inoculation:
With material in (2) on inoculating paper, aseptic blade and terminal bud and the base portion 0.5cm stem section of cutting, every bottle graft 1-3 section is put into induced dormancy under 24 ℃ ± 1 ℃ dark condition of culture, checks pollution condition every other day, rescues untainted;
(5) after cultivating in 35-45 days, sleeping bud will be induced on the culture in (4) successively, move on to 2000Lx under the light training condition, illumination in 16 hours, when the isometric 0.5-1cm of arriving grows, cut sleeping bud, be put in the 150ml triangular flask, every bottle of 5-7 point, add M2 to 75ml line, be put into the swinging shaking table that per minute 80 changes, through about 45-50 days, grow embryoid (plumule) successively, per 10 days subcultures once, until covering with one deck, along with embryoid " ball " increases, manually cut brokenly spheroid with inoculator every now and then, make it accelerate propagation;
(2) shaking table level liquid amplification cultivation:
(1) collect the embryoid of cultivating in the aforesaid liquid (plumule), transfer to the 500ml triangular flask, add M2 to 200ml line from the little triangular flask of 150ml, per 10 days subcultures once, during each subculture by enlarging colony at 1: 2;
(2) mensuration of embryo shape technical parameter:
At 500ml one-level cultivation stage, must finish the mensuration of following several technical parameters, could determine the cultivation system of industrialization production usefulness.
A. the mensuration of " than biological growth speed ": (hereinafter to be referred as " growth rate ") higher plant is cultivated system's (cell, embryoid etc.) to reach industrialized requirement, it is believed that the minimum 5.7mg/gd of its growth rate, it is every milliliter of cultivation, increase the 5.7mg fresh weight every day, industrialization just is of practical significance.This technical parameter is measured and is applied in industrialization production overall process, constantly measures, constantly screening.Our present cultivation system is stabilized in the 7.2-8.5mg/gd level.
B. assay method and computing formula:
The bright product of each 500ml triangular flask inoculation 15g (the inoculation size influences the result), liquid feeding 200ml (weight g)
10 days (determining according to Dendrobium officinale polysaccharide content requirement and proliferative amount decision of 10 day cycle) claims fresh weight behind the subculture.
C. result of calculation then:
Results fresh weight-15g ÷ 200 ÷ 10=mg/gd
Dendrobium officinale polysaccharide is measured: regularly (by Various Seasonal, by making a gesture of measuring) send professional authority mechanism to detect, and this cultivation polyoses content is stabilized in 8-11% at present, is the 85-90% of 3 years living plant content of artificial cultivation;
D. the rate of must doing is measured: each ripe cultivation ties up to after each results, after drench is done, claims fresh weight, obtains dry weight after the oven dry; Calculate: dry weight ÷ fresh weight=%, our rate of must doing is 9% ± 1%;
(3) no hormone is cultivated the screening (carrying out at 500ml one-level cultivation stage) of system:
(1) selection is above-mentioned on the M2 medium, and the energy optimum state satisfy above-mentioned several technical parameters and cultivate system as the starting material that screens, every bottle graft kind 15g fresh weight, adding M0 (M2 removes exogenous hormone, and all the other are constant);
(2) once, behind 5-8 successive transfer culture of process, enter (two) (2) and detect every technical parameter program equally, select to meet the starting material of the cultivation system of parameters as the next stage cultivation with 10 days subcultures;
(4) no hormone secondary aerobic culture:
(1) the maturation system of receipts (three) (2) with 1 pair 1 vaccination ways, is transferred to the 1000ml triangular flask together from the culture and the conditioned medium (former female bottle medium) of 500ml triangular flask cultivation after 10 days, adds fresh M0 medium to the 800ml line;
(2) above-mentioned 1000ml triangular flask, load onto 0.45 μ aperture is housed, diameter 25mm air filtering, by per two 1000ml triangular flasks is one group parallel connection, and promptly an intake air filter is responsible for two bottles, the air exit of two bottles of a discharge filter burden, connect 5 watts of ventilation pumps then, the air inlet bottom connects, fish jar ventilation sand head, 16 1000ml triangular flasks of each pump burden; After the ventilation, illumination in 14-16 hour is provided every day or takes sun natural daylight, per 20 days cultivation cycle were cultivated 15～20 days;
(5) the ultimate cultivation of no hormone (finished product is cultivated and produced):
For contaminated solution and pollution cause seed costs, select 20 liters, ready-made barreled bucket is as ultimate culture vessel;
The ultimate liquid amount of each container is 16 liters, wherein 14 be upgraded to fresh M0 liquid, the conditioned medium (former female bottle medium) that 2 are upgraded to (four) when 2. cultivating is made seed with the culture of (four) 6 1000ml triangular flasks 2., after the inoculation, with (four) air-breather 2., each 5 watts of ventilation pump, the throughput of responsible 4 20L containers keeps the 2000-2500Lx luminous intensity, press 38-40 degree inclined-plane on autocratic culturing rack, each shelf takes up an area of 1M
2, adorn three layers, 3 20L containers of a row, each shelf is placed 18, stops after 20 days cultivating, and each container can be gathered in the crops the bright product of 3.2-3.7 kilogram;
(6) results of dried finished product sterilization:
The aquatic foods product of gathering in the crops from the 20L container, behind purified rinse water one time, drench is done or was blown 2-3 hour with electric fan, then, is distributed into thin layer, is put in the exhaust bakeout case 55 ℃-58 ℃, disposable oven dry.
Dry product is pressed 250g, 500g, 1000g, 5000g different basis weights packing, and collection is contained in the carton respectively, behind γShe Xianmiejun, preserves as ready for 8 ℃-12 ℃.
Below be the concrete composition tabulation of medium related among the present invention:
Claims (8)
1. the method for a hormone-free scale culture and production of dendrobium officinale embryo dry product comprises:
(1) cultivation of explant and inoculation
Dendrobium candidum original seed plant is cultivated into the 3-5cm height, band 3-5 sheet leaf, the adult strain of tool 3-4 internode, gather explant and wash repeatedly till the non-foam,, cut full leaf and plant base portion 0.3～0.6cm then with its sterilization with running water, be inoculated into the M2 solid culture medium, under dark condition of culture, 24 ℃ ± 1 ℃, the growth of induced dormancy bud 35～45 days;
(2) shoot proliferation of embryoid
After inducing sleeping bud successfully, move on to 2000Lx under the light training condition, sleeping bud is peeled off in illumination in 16～20 hours when growing to 0.8～1.2cm, be moved into the M2 liquid nutrient medium, per 7 days subcultures once, 24 ℃ ± 1 ℃, nature room light intensity after 45-50 days, induces embryoid;
(3) no hormone one-level is cultivated and screening
Get above-mentioned embryoid and be inoculated into the M2 liquid nutrient medium, carry out the level liquid enlarged culture, per 10 days subcultures once, each subculture enlarged colony by 1: 2, through 3-5 all after date, selected that the form growth is normal, green, the big or small close cultivation of profile system, use the M0 liquid nutrient medium, per 10 days subcultures once carry out 5～8 times successive transfer culture, get mature cell system;
(4) no hormone secondary aerobic culture
With above-mentioned mature cell is to be inoculated into the M0 liquid nutrient medium to carry out secondary ventilation enlarged culture, and adds the conditioned medium after one-level is cultivated, and illumination in 14-16 hour is provided every day or takes sun natural daylight, cultivates 15～20 days;
(5) the ultimate aerobic culture of no hormone
Select culture vessel, each container liquid amount is 16 liters, wherein 14 be upgraded to fresh M0 liquid nutrient medium, the conditioned medium that 2 are upgraded to step (4) when cultivating, the seed of selecting step (4) to obtain is inoculated, keep the 2000-2500Lx luminous intensity, on autocratic culturing rack, cultivated 15～20 days, collect bright product by 38-40 degree inclined-plane;
(6) post processing of bright finished product
After above-mentioned bright product collection, do, weigh with drench behind the purified rinse water one time, then in 50～60 ℃ of dryings, press 250g, 500g, 1000g or 5000g quantitative package, behind γShe Xianmiejun, 8 ℃ of-12 ℃ of preservations.
2. the method for a kind of hormone-free scale culture and production of dendrobium officinale embryo dry product according to claim 1; it is characterized in that: the M2 solid culture medium described in the step (1) is 1/2MS macroelement+organic routine of full dose molysite+full dose trace+full dose+sucrose 30g/L+ caseinhydrolysate CH 500mg/L+ α-Nai Yisuan NAA2mg/L+ basic element of cell division BA0.2mg/L+100g bananas juice/L+ agar powder 4.8g/L; PH5.8, conventional sterilization.
3. the method for a kind of hormone-free scale culture and production of dendrobium officinale embryo dry product according to claim 1; it is characterized in that: the sterilization described in the step (1) is after 12-15 minute → aqua sterilisa washes several times in 50% ethanol infiltration 3-5 second → 20% " 84 " liquid 7-8 minute → 0.1% mercuric chloride liquid, suck dry moisture.
4. the method for a kind of hormone-free scale culture and production of dendrobium officinale embryo dry product according to claim 1; it is characterized in that: the M2 liquid nutrient medium described in step (2) and (3) is 1/2MS macroelement+organic routine of full dose molysite+full dose trace+full dose+sucrose 30g/L+ caseinhydrolysate CH500mg/L+ α-Nai Yisuan NAA2mg/L+ basic element of cell division BA0.2mg/L+100g bananas juice/L; PH5.8, conventional sterilization.
5. the method for a kind of hormone-free scale culture and production of dendrobium officinale embryo dry product according to claim 1; it is characterized in that: the M0 liquid nutrient medium described in the step (4) is the 1/2MS macroelement+organic routine of full dose molysite+full dose trace+full dose+sucrose 30g/L; PH5.8, conventional sterilization.
6. the method for a kind of hormone-free scale culture and production of dendrobium officinale embryo dry product according to claim 1, it is characterized in that: the conditioned medium described in the step (4) is the old medium after cultivating 10～15 days in the M0 liquid nutrient medium of the stem of noble dendrobium in step (3).
7. the method for a kind of hormone-free scale culture and production of dendrobium officinale embryo dry product according to claim 1, it is characterized in that: the conditioned medium described in the step (5) is the old medium after cultivating 10～15 days in the M0 liquid nutrient medium of the stem of noble dendrobium in step (4).
8. the method for a kind of hormone-free scale culture and production of dendrobium officinale embryo dry product according to claim 1 is characterized in that: the autocratic culturing rack described in the step (5) is that each shelf takes up an area of 1M
2, adorn three layers, 3 20L containers of a row, each shelf is placed 18.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102266492A (en) * | 2011-07-22 | 2011-12-07 | 安徽康顺名贵中草药产业开发有限公司 | Method for processing dendrobium huoshanense plant dry product |
| CN103385174A (en) * | 2013-07-17 | 2013-11-13 | 宁波神乙草生物科技有限公司 | Tissue culture and inoculation method for dendrobium officinale |
| CN103460928A (en) * | 2013-09-05 | 2013-12-25 | 深圳市农科集团有限公司 | Method for proliferating and propagating dendrobium |
| CN105851385A (en) * | 2016-05-03 | 2016-08-17 | 泉州正和堂生物科技有限公司 | Dendrobium nobile monomer germ tea and preparation method thereof |
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2009
- 2009-05-21 CN CN2009100516635A patent/CN101558740B/en active Active
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102266492A (en) * | 2011-07-22 | 2011-12-07 | 安徽康顺名贵中草药产业开发有限公司 | Method for processing dendrobium huoshanense plant dry product |
| CN103385174A (en) * | 2013-07-17 | 2013-11-13 | 宁波神乙草生物科技有限公司 | Tissue culture and inoculation method for dendrobium officinale |
| CN103385174B (en) * | 2013-07-17 | 2015-11-18 | 宁波神乙草生物科技有限公司 | Candidum tissue culturing inoculation method |
| CN103460928A (en) * | 2013-09-05 | 2013-12-25 | 深圳市农科集团有限公司 | Method for proliferating and propagating dendrobium |
| CN103460928B (en) * | 2013-09-05 | 2015-09-09 | 深圳市农科集团有限公司 | Stem of noble dendrobium propagation expanding propagation method |
| CN105851385A (en) * | 2016-05-03 | 2016-08-17 | 泉州正和堂生物科技有限公司 | Dendrobium nobile monomer germ tea and preparation method thereof |
| CN115644058A (en) * | 2022-09-23 | 2023-01-31 | 广西壮族自治区农业科学院 | Method for promoting orchidaceae plant seeds to quickly grow seedlings |
| CN115644058B (en) * | 2022-09-23 | 2023-11-21 | 广西壮族自治区农业科学院 | Method for promoting quick seedling formation of orchid seeds |
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