CN105145351B - The method of candidum tissue culturing forming seedling through one step culture mass production - Google Patents
The method of candidum tissue culturing forming seedling through one step culture mass production Download PDFInfo
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Abstract
The invention discloses the method for candidum tissue culturing forming seedling through one step culture mass production, comprise the following steps：(1) breeding of protocorm：The terminal bud or axillary bud for selecting excellent iron sheet dendrobium plant are explant, after explant sterilization on MS+6 BA0.1~3.0mg/L+KT0.2~1.0mg/L+IAA0.1~1.0mg/L+NAA0.2~2.0mg/L+ cycocel 0.01~0.5mg/L Solid agar cultures, healthy and strong bud is differentiated while induction protocorm；(2) forming seedling through one step culture mass production：Directly being transferred on the Solid agar culture of MS+6 BA0.2~1.0mg/L+IAA0.2~0.5mg/L+NAA0.1~0.5~2.0mg/L of 2.0mg/L+ cycocels with bud protocorm of obtaining of step (1) is bred, broken up, strong sprout and is taken root；(3) test tube transplantation of seedlings.The method for tissue culture of short, low cost dendrobium candidum efficient low-consume amount the invention provides a kind of breeding cycle；Industrialized production is especially suitable for, and reaches industrialization level.
Description
Technical field
The invention belongs to the tissue culture technology field of dendrobium candidum, and in particular to a kind of candidum tissue culturing seedling forming seedling through one step culture batch
The method for quantifying production.
Background technology
Dendrobium candidum (Dendrobium officinale Kimura et Migo) is the orchid family stem of noble dendrobium, belongs to perennial herb
Plant.The medicinal and health value of dendrobium candidum is high, and " dendrobium candidum " one word begins to come from《Sheng Nong's herbal classic》, international medicinal plant
Thing circle is called " medicine circle giant panda "；It is referred to as " help celestial grass " by among the people.Modern clinic application and pharmacological research confirmation, iron sheet stone
Dry measure used in former times has nourishing Yin and moistening lung, nourishing the stomach to improve the production of body fluid, brain nourishing and eyesight improving, liver protection calming heart, heat-clearing and fire-reducing, the consumptive disease of tonifying five zang organs, the god of macrobiosis of making light of one's life by commiting suicide
Outstanding service is imitated.To chronic fatigue syndrome, tuberculosis, coronary heart disease, ephrosis, chronic liver disease, hypertension, high fat of blood, hyperglycaemia, glycosuria
The diseases such as disease, chronic gastropathy, dry syndrome, cancer have definite curative effect and conditioning rehabilitative action.Can strengthen body immunity,
Anti-oxidant, anti-aging, to excessive drinking and smoking, overwork, night life excessively, with eye is overstrain one's nerves, hoarseness, mouth parched and tongue scorched etc.
Rehabilitation has special efficacy.
The seed of dendrobium candidum is minimum, without endosperm, every seed only have under 3-4 cell, natural conditions need it is true with some
Bacterium symbiosis could sprout, it is difficult to be cultivated with seedling；And the breeding potential of the mode such as traditional plant division, cuttage is extremely low, add artificial
Excessive excavation and ecological environment destruction, its resource is endangered.Dendrobium candidum tissue culture quick breeding technology is solution
A certainly maximally effective approach of dendrobium candidum commerial growing seedling.
In recent years, to dendrobium candidum tissue culture, quickly breeding has carried out a large amount of industrialization technology researchs to researcher,
But major part is using explant-protocorm induction-differentiation-strong sprout -- the approach taken root-transplant carries out factorial praluction,
Culture program is especially cumbersome；In addition, culture medium need to add special composition, the healthy and strong seedling of high-quality could be obtained.Such as newest iron
Skin stem of noble dendrobium Chinese patent 201310681992.4,201310699964.5,201410113720.9 etc., except minimal medium and swash
It is plain outer, also need addition bananas juice or potato juice or proline or caseinhydrolysate or Tea Polyphenols or activated carbon etc. other
Composition, could obtain the test tube seedling of stalwartness, so that in the market candidum tissue culturing seedling price is high, at least want 15 yuan/bottle.Plantation
Family be cannot afford high quality seedling, so as to constrain dendrobium candidum high quality seedling Industry Promotion.Therefore, this invention address that exploitation one
Plant cultivating system efficiently, inexpensive.
The content of the invention
For dendrobium candidum tissue cultures culture program is cumbersome, high cost problem, it is an object of the present invention to provide one kind
Breeding cycle is short, low cost dendrobium candidum efficient low-consume amount method for tissue culture.The inventive method is especially suitable for industrialization
Production, and reach industrialization level.
The purpose of the present invention is achieved in the following ways：
The method of candidum tissue culturing forming seedling through one step culture mass production, comprises the following steps：
(1) breeding of protocorm：The terminal bud or axillary bud of excellent iron sheet dendrobium plant are selected for explant, after explant sterilization
In MS+6-BA0.1~3.0mg/L+KT0.2~1.0mg/L+IAA0.1~1.0mg/L+NAA0.2~2.0mg/L+ cycocels
On 0.01~0.5mg/L Solid agar cultures, healthy and strong bud is differentiated while induction protocorm；
(2) forming seedling through one step culture mass production：By step (1) obtain band bud protocorm be directly transferred to MS+6-BA0.2~
On the Solid agar culture of 1.0mg/L+IAA0.2~0.5mg/L+NAA0.1~0.5~2.0mg/L of 2.0mg/L+ cycocels
Bred, broken up, strong sprout and being taken root；
(3) test tube transplantation of seedlings.
In step (1) by explant be cut into 0.5-1cm it is long containing 11 bud of section (i.e. at least containing a terminal bud or axillary bud
Stem section) specification.
Step (1) explant disinfecting process：Cleaned up with washing powder first, with aseptic washing 5 on aseptic operating platform
~7 times, infiltrated 30 seconds~1 minute with 70% alcohol, place into 0.1% mercuric chloride solution and sterilize 8~15 minutes, sterilized water punching
Wash 5~6 times, it is standby.
The culture medium that step (1) is preferably used：MS+6-BA2.0mg/L+KT0.5mg/L+IAA0.25mg/L+
NAA0.5mg/L+ cycocels 0.1mg/L；Or preferably use culture medium：MS+6-BA1.5mg/L+KT0.5mg/L+
IAA0.3mg/L+NAA0.4mg/L+ cycocels 0.3mg/L.
The protocorm that step (1) is induced continues squamous subculture, every 40~50d subcultures once.
The culture medium that step (2) is preferably used：MS+6-BA0.3mg/L+IAA0.3mg/L+NAA0.4mg/L+ cycocels
0.6mg/L；Or preferably use culture medium：MS+6-BA0.5mg/L+IAA0.2mg/L+NAA0.6mg/L+ cycocels 0.9mg/
L。
Step (2) is emerged once when cultivating every 35~45d.
The cultivation temperature of step (1) and (2) is 24 DEG C ± 2, in 1000~2000lx environment, daily illumination in 10~12 hours
Culture.
Step (3) test tube transplantation of seedlings detailed process：Before test tube seedling bottle outlet is transplanted, by the bottle seedling that root leaf is various, place outdoor
Hardening 5-7d, then takes out cleaning root culture medium, by seedling replanting to cultivation matrix, mass ratio tongue in cultivation matrix
Moss：Bark:Vermiculite=5:3:1, it is 20~28 DEG C in temperature, grown under conditions of air humidity 80~90%, 30~40d grows
During young leaves, that is, survive.
The present invention compared with prior art, the beneficial effects of the present invention are：
1st, in place of in view of the shortcomings of the prior art, the present invention obtains a large amount of protocorms in a short time by tissue culture technique
Stem, proliferation times reach 22-45, and general proliferation times are between 5-20；The protocorm of existing tissue culture method growth is general all
Cut, and the protocorm then obtained using the inventive method and culture medium is without cutting, direct rolling bottle (can be as seen in Figure 2
The one plant of single protocorm of strain for growing, without being separated using instrument), method is extremely simple, can save inoculation work
Production cost.
2nd, according to the research of forefathers, the tissue cultures of dendrobium candidum mainly use the protocorm of explant one (Fiber differentiation)
Differentiation seedling (differentiation culture) strong sprout (strong seedling culture) such as is taken root (culture of rootage)-transplants (transplant culture) at multiple the culture
Program is completed, and at least needs 120d.Culture program of the present invention is completed in two steps, and Protocorm Multiplication multiple is improved first, accelerates base
The breeding (proliferation times reach 22-45) of plinth seedling, realizes large-scale production within a short period of time；Secondly, the mass production stage is former
Bulb synchronization and is taken root at Proliferation, Differentiation strong sprout, and four steps in culture program are reduced into a step completes, i.e. " forming seedling through one step culture " method, shape
Only needed 35-45 days into whole plant, having saved 60~80d times obtains high quality seedling, and production efficiency is high.
3rd, during Protocorm Multiplication, low concentration cycocel (concentration is 0.01-0.5mg/L) is added, promotes the propagation of protocorm
And differentiation, and sprout is healthy and strong；During mass production, then add higher concentration cycocel and cultivated, can obtain high-quality
Seedling.The present invention in process of production, according to the growing way of seedling, controls the suitable concentration of cycocel, can obtain optimal culture effect
Really.
4th, as a example by producing 10000L dendrobium officinale culture mediums, such as every liter addition 100g banana need to add 1000kg bananas,
Unit price is 7.0 yuan/kg, increased 7000 yuan of cost；Such as every liter of addition 1g proline, 10kg proline is needed, 1.8 yuan of unit price/
G, increased 18000 yuan of cost；100 milliliters of coconut milk of every liter of addition, need 1000000ml coconut milk, unit price for 0.015 yuan/
Ml, increased 15000 yuan of cost.The conventional MS of present invention selection is minimal medium, and cycocel is strengthening agent, fragrant without addition
Other special compositions such as any of several broadleaf plants juice, potato juice, proline, caseinhydrolysate, Tea Polyphenols, activated carbon；The inventive method is configured
10000L culture mediums, only need 5-10 grams of cycocel, and unit price is 0.9 yuan/g, it is only necessary to 4.5-9.0 units, negligible.The present invention is not
Sterile working step is only reduced, and has saved culturing room space, simplify health seedling production routine, considerably reduced
Production cost, improves production efficiency.
Brief description of the drawings
Fig. 1 is dendrobium candidum Protocorm Multiplication differential growth situation；
Fig. 2 is the scattered protocorm of dendrobium candidum；
Fig. 3 is the situation after dendrobium candidum protocorm rolling bottle；
Fig. 4 is dendrobium candidum squamous subculture protocorm synchronization Proliferation, Differentiation strong sprout and situation of taking root；
Fig. 5 Proliferation, Differentiation strengthening seedling and rooting seedlings；
Fig. 6 is the dendrobium candidum test tube seedling growing state of transplant survival；
Fig. 7 is the extensive bottle seedling production of dendrobium candidum；
Fig. 8 is the extensive basin seedling production of dendrobium candidum；
Fig. 9 is the extensive seedling production of dendrobium candidum.
Specific embodiment
The present invention is intended to further illustrate with reference to embodiments, is not intended to limit the present invention.
Embodiment 1, tissue-culturing quick-propagation dendrobium candidum.
First, culture medium is prepared and sterilized
Initial culture base is used for basic seedling breeding：MS+6-BA2.0mg/L+KT0.5mg/L+IAA0.25mg/L+
NAA0.5mg/L+ cycocels 0.1mg/L
Subculture medium is used for forming seedling through one step culture mass production：MS+6-BA0.3mg/L+IAA0.3mg/L+NAA0.4mg/L
+ cycocel 0.6mg/L.
At 121 DEG C of above culture medium, sterilize 20 minutes.
2nd, basic seedling breeding
The dendrobium candidum 0.5cm stem sections with terminal bud long are taken, is cleaned up with washing powder first, nothing is used on aseptic operating platform
Bacterium is washed 5~7 times, is infiltrated 1 minute with 70% alcohol, places into 0.1% mercuric chloride solution and sterilizes 8~12 minutes, sterilized water
Rinse 5~6 times, terminal bud and axillary bud are transferred to equipped with Initial culture base, every bottle of explant number is 2~3, in following cultures
Under the conditions of cultivated：24 DEG C or so, daily illumination in 10 hours, intensity of illumination is 1000~2000lx or so；Terminal bud inoculation 30
Individual, axillary bud is inoculated with 50.Culture 8 weeks or so, obtains 125 protocorms.When basic seedling is bred, protocorm is transferred to Initial culture base
In, every 45d or so propagation once, in each subculture, Protocorm Multiplication multiple average out to 30, up to 42.It is former with one-step inducing
Bulb and bud growing state, as shown in Figure 1.According to production scale, sufficient basic seedling is bred, then carry out mass production.
3rd, forming seedling through one step culture mass production
The 10000 strain protocorms taken in above-mentioned 125 strains carry out mass production, and specific method is as follows：Will step
It is rapid two obtain protocorms as shown in Fig. 2 being directly transferred to one-step culture base in cultivated, as shown in figure 3, condition of culture
Same step 2；After culture 35-45d, each protocorm breaks up budlet from base portion simultaneously, gradually grows up to 2-6cm healthy and strong clump buds high,
And the whole plant of 5-10 bar roots is grown from base portion.Synchronous Proliferation, Differentiation strong sprout and take root test tube seedling growing state such as Fig. 4,
Shown in Fig. 5.
4th, test tube transplantation of seedlings：
Before test tube seedling bottle outlet is transplanted, by the bottle seedling that root leaf is various, outdoor hardening 5-7d is placed, then take out the training of cleaning root
Base is supported, is 20~28 DEG C in temperature by seedling replanting to cultivation matrix, grown under conditions of air humidity 80~90%, 30
When~40d grows young leaves, already survive, as shown in Figure 6.
Dendrobium candidum tissue culture propagation method provided by the present invention, terminal bud or stem section are explant, and materials are easy, number
Amount is big, and genetic stability is high, the advantage such as well-grown.During Initial culture, with one-step inducing protocorm and bud, breeding coefficient
Greatly.Mass production stage, the dendrobium candidum cultural method unified using four steps simplifies health seedling production routine.With life
Produce as a example by 10000L dendrobium officinale culture mediums, such as every liter addition 100g banana need to add 1000kg bananas, unit price for 7.0 yuan/
Kg, increased 7000 yuan of cost；Such as every liter of addition 1g proline, 10kg proline, 1.8 yuan/g of unit price is needed to increased 18000
The cost of unit；100 milliliters of coconut milk of every liter of addition, need 1000000ml coconut milk, and unit price is 0.015 yuan/ml, increased 15000
The cost of unit.It is strengthening agent that the present invention uses cycocel, configures 10000L culture mediums, it is only necessary to which 5-10g cycocels, price is extremely just
Preferably, cost is negligible.The present invention not only reduces sterile working step, and has saved culturing room space, simplifies strong
Health seeling industry program, reduces production cost, improves production efficiency.The technology of the present invention has been successfully applied to large-scale production
In, and extraordinary application effect is obtained, as shown in Fig. 7, Fig. 8, Fig. 9.
Embodiment 2, tissue-culturing quick-propagation dioscorea zingiberensis wright
First, culture medium is prepared and sterilized
2nd, Initial culture base：MS+6-BA1.5mg/L+KT0.5mg/L+IAA0.3mg/L+NAA0.4mg/L+ cycocels
0.3mg/L；
3rd, subculture medium：MS+6-BA0.5mg/L+IAA0.2mg/L+NAA0.6mg/L+ cycocels 0.9mg/L
At 121 DEG C of above culture medium, sterilize 20 minutes.
Take dendrobium candidum (Dendrobium officinale Kimura et Migo) terminal bud or stem section, basic operation side
Method is consistent with embodiment 1；Effect is more or less the same with embodiment 1.
Claims (7)
1. the method for candidum tissue culturing forming seedling through one step culture mass production, it is characterised in that comprise the following steps：
(1) breeding of protocorm：The terminal bud or axillary bud of excellent iron sheet dendrobium plant are selected for explant, in MS after explant sterilization
+ 6-BA2.0mg/L+KT0.5mg/L+IAA0.25mg/L+NAA0.5mg/L+ cycocels 0.1mg/L；Or MS+6-BA1.5mg/L+
On KT0.5mg/L+IAA0.3mg/L+NAA0.4mg/L+ cycocel 0.3mg/L Solid agar cultures, the same of protocorm is induced
When differentiate healthy and strong bud；
(2) forming seedling through one step culture mass production：The band bud protocorm that step (1) is obtained directly is transferred to MS+6-BA0.3mg/L+
IAA0.3mg/L+NAA0.4mg/L+ cycocels 0.6mg/L；Or MS+6-BA0.5mg/L+IAA0.2mg/L+NAA0.6mg/L+
Bred on the Solid agar culture of cycocel 0.9mg/L, broken up, strong sprout and being taken root；
(3) test tube transplantation of seedlings.
2. the method for candidum tissue culturing forming seedling through one step culture mass production according to claim 1, it is characterised in that step
(1) explant is cut into the 0.5-1cm specifications containing 11 bud of section long in.
3. the method for candidum tissue culturing forming seedling through one step culture mass production according to claim 1 and 2, it is characterised in that
Explant disinfecting process：Cleaned up with washing powder first, with aseptic washing 5~7 times on aseptic operating platform, use 70% alcohol
Infiltration 30 seconds~1 minute, places into 0.1% mercuric chloride solution and sterilizes 8~15 minutes, and aseptic water washing 5~6 times is standby.
4. the method for candidum tissue culturing forming seedling through one step culture mass production according to claim 1, it is characterised in that step
(1) protocorm for inducing continues squamous subculture, every 40~50d subcultures once.
5. the method for candidum tissue culturing forming seedling through one step culture mass production according to claim 1, it is characterised in that step
(2) emerged once every 35~45d when cultivating.
6. the method for candidum tissue culturing forming seedling through one step culture mass production according to claim 1, it is characterised in that step
(1) and (2) cultivation temperature be 24 DEG C ± 2 DEG C, in 1000~2000lx environment, daily 10~12 hours illumination cultivations.
7. the method for candidum tissue culturing forming seedling through one step culture mass production according to claim 1, it is characterised in that step
(3) test tube transplantation of seedlings detailed process：Before test tube seedling bottle outlet is transplanted, by the bottle seedling that root leaf is various, outdoor hardening 5-7d is placed, then
Take out cleaning root culture medium, by seedling replanting to cultivation matrix, mass ratio liver moss in cultivation matrix：Bark:Finger stone
=5:3:1, it is 20~28 DEG C in temperature, grown under conditions of air humidity 80~90%, when 30~40d grows young leaves, i.e.,
Survive.
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Families Citing this family (4)
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| CN106386482B (en) * | 2016-08-31 | 2018-06-08 | 南京仙草堂生物科技有限公司 | Dendrobium candidum high quality seedling nature optical culture once-seedling forming quick propagating method |
| CN109644871B (en) * | 2019-01-21 | 2022-01-11 | 花垣县精虹生物科技发展有限公司 | Batch production method suitable for various orchidaceous medicinal plants |
| CN115250915B (en) * | 2022-08-12 | 2023-05-26 | 云南中医药大学 | Efficient propagation method for inducing protocorm and plant regeneration by stem segments of dendrobium nobile |
| CN116965335B (en) * | 2023-09-05 | 2024-09-10 | 河北农业大学 | Tissue culture and rapid propagation method for dendrobium candidum |
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