CN107232061B - A kind of special culture media and tissue culture method of rapid induction bletilla bulb - Google Patents
A kind of special culture media and tissue culture method of rapid induction bletilla bulb Download PDFInfo
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- CN107232061B CN107232061B CN201710552200.1A CN201710552200A CN107232061B CN 107232061 B CN107232061 B CN 107232061B CN 201710552200 A CN201710552200 A CN 201710552200A CN 107232061 B CN107232061 B CN 107232061B
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- 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
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- 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
- A01H4/008—Methods for regeneration to complete plants
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Abstract
The invention discloses a kind of special culture medias of rapid induction bletilla bulb, the special culture media is efficiently induced by bletilla seed germination medium and bulb, strong seedling culture base forms, wherein: the formula of the bletilla seed germination medium are as follows: 1/2MS, NAA0.1mg/L, GA30.1mg/L, agar 6.5-7.0g/L, white sugar 20g/L, pH5.8-6.0；Described and bulb efficiently induces, the formula of strong seedling culture base are as follows: 1/2MS, NAA0.5~0.6mg/L, 6-BA0.6~0.8mg/L, PP3330.4~0.5mg/L, white sugar 30-45g/L, 25~40g/L of banana puree, active carbon 0.1g/L, pH5.8~6.0.The invention also discloses the tissue culture methods using the special culture media.This method simplifies the production link of bletilla pseudobulb tissue-cultured seedling, induce pseudobulb, pseudobulb fast-growth, and Rooting and hardening-off culture is completed in same step, for in 90 days the time required to from seed to transplanting, shorten incubation time, the fast-growth for promoting pseudobulb differentiation rate, pseudobulb improves the transplanting survival rate of tissue-cultured seedling.
Description
Technical field
The invention belongs to bletilla technical field of cultivation, and in particular to a kind of special culture media of rapid induction bletilla bulb and
Tissue culture method.
Background technique
Bletilla is that (Bletilla striata (Thunb.) Reichb.f., pseudobulb are medicinal portion to orchid bletilla
Position, have effects that astringing to arrest bleeding, detumescence and promoting granulation, now research discovery bletilla treatment pulmonary tuberculosis, stomach trouble, in terms of effect
Obviously, while still good chemical material.But bletilla plant is destroyed by artificial excavation, raw mirror, and wild resource is very rare,
It has been listed in rare plant second class protection kind.
With the development need of bletilla value of exploiting and utilizing, market demand is continuously increased, and wild resource ten
Dividing rareness, breeding potential is lower under bletilla natural conditions, so be badly in need of carrying out bletilla artificial propagation and the research of standardized cultivation,
To preferably protect bletilla wild resource, and ensure the market demand and development and utilization of bletilla.
Under the conditions of traditional cultivation, for bletilla based on division propagation, reproduction speed is slow, and breeding coefficient is extremely low, and is easy to lead
The accumulation and propagation for evil of causing a disease, it is difficult to the needs of suitable for modern plantation.And seed amount is big in bletilla fruit pod, but because seed knot
Structure is special, is difficult to sprout under natural conditions, so the rare bottleneck as its large-scale development of bletilla seedling.And with biological skill
The development of art, bletilla tissue culture technology have obtained very fast development, generally all pass through aseptic seeding, tufted seedling culture, strong seedling culture, life
Multiple links such as root culture, and work consuming, time-consuming, tissue-cultured seedling bulb differentiation rate is not high under normal conditions, the transplant survival of tissue-cultured seedling
Rate is not also high, to largely affect the production application of bletilla tissue-cultured seedling.So how to simplify tissue culture link, reduce at
This, promotes the differentiation of bulb, fast-growth to become an important factor for influencing tissue-cultured seedling transplanting survival rate and production application.
201110119312.0 also disclosing the method and its dedicated culture for preparing bletilla pseudobulb in a kind of culture vessel
Base, which describe using bletilla seed as the special culture media of explant, by seed germination medium, pseudobulb induced medium,
Pseudobulb proliferated culture medium and pseudobulb root media composition.Each of which step needs a specific culture medium, training
Feeding mode has the defects of work consuming, time-consuming as described above.
Summary of the invention
For the defect of existing bletilla bulb tissue culture technology, the purpose of the present invention is to provide a kind of rapid induction bletilla squamas
The special culture media and tissue culture method of stem, the method is easy to operate, and production cost is low, and effect stability.
In order to achieve the above objectives, the invention provides the following technical scheme:
1, a kind of special culture media of rapid induction bletilla bulb, the special culture media is by bletilla seed germination medium
It is efficiently induced with bulb and strong seedling culture base forms, in which:
The formula of the bletilla seed germination medium are as follows: 1/2MS, NAA0.1mg/L, GA30.1mg/L, agar 6.5~
7.0g/L, white sugar 20g/L, pH5.8~6.0；Described and bulb efficiently induces and the formula of strong seedling culture base are as follows: 1/2MS,
NAA0.5~0.6mg/L, 6-BA0.6~0.8mg/L, PP3330.4~0.5mg/L, 30~45g/L of white sugar, banana puree 25~
40g/L, active carbon 0.1g/L, pH5.8-6.0.
Preferably, the bulb efficiently induces and the formula of strong seedling culture base are as follows: 1/2MS, NAA0.6mg/L, 6-
BA0.8mg/L, PP3330.5mg/L, white sugar 40g/L, banana puree 25g/L, active carbon 0.1g/L, pH5.8-6.0.
2, a kind of tissue culture method of rapid induction bletilla bulb, which comprises the steps of:
1) surface the sterilization of bletilla seed: is washed into the alcohol painting that uncracked bletilla seed mass fraction is 75%
Fruit pod, is then placed in 0.1% mercuric chloride 15~20min of immersion by 1-3min on superclean bench, then with aseptic water washing 3
~5 times, aseptic filter paper blots surface moisture in case inoculation；
2) under sterile operating condition, the bletilla seed of step 1) sterilization is inoculated into described in claim a kind
It is cultivated in sub- germination medium；
3) bulb height described in claim 1 is forwarded to when step 2) the bletilla seed sprouts 0.8~1.2cm of height of seedling
It is cultivated in effect induction and strong seedling culture base.
It is further preferred that the condition of culture of step 2) are as follows: illumination 1500~2000Lx, 10~12h/ days；Temperature, 24~
28℃。
It is further preferred that the condition of culture of step 3) are as follows: illumination 1500~2000Lx, 12~15h/ days；Temperature, 24~
28℃。
Further, the acquisition of bletilla seed described in the above method and storage practice are as follows: acquisition 9-10 month bletilla is mature, full
Full fruit pod is placed in the place of shady and cool ventilation, and spreading for cooling 2~3 weeks；When fruit pod epidermis becomes light brown, fruit pod is loaded on kraft paper bag
In be stored in 4 DEG C of refrigerating chambers of refrigerator, it is ensured that used in bletilla tissue-cultured seedling whole year production.
NAA used in the present invention is methyl α-naphthyl acetate, GA3For gibberellin, 6-BA is 6- benzyl aminoadenine, PP333For multiple-effect
Azoles, reagent are that analysis is pure, and water is ultrapure water.
The beneficial effects of the present invention are: then this method is used using promoting seed quick-speed germination to cultivate as the first stage
Efficient culture medium adjusts the methyl α-naphthyl acetate of Furcation defects, growth that is, to promote the hormone 6- benzyl aminoadenine quickly broken up, and
The paclobutrazol for adjusting plant hormone balance, improving resistance, three kinds of hormones are formula, screen suitable concentration and ratio, make it
The differentiation and growth of preferable regulation bletilla tissue culture vacation seedling bulb, cauline leaf, root；And be added the suitable nutrition original-white sugar of concentration and
Banana puree has effectively promoted the fast-growth of bulb, and active carbon appropriate can preferably promote the growth of root, and can be effective
The some toxin generated in absorption bletilla growth.So the cultural method simplifies the production link of bletilla pseudobulb tissue-cultured seedling,
Collect induction pseudobulb, pseudobulb fast-growth and Rooting and hardening-off culture to complete in a step, is 90 the time required to from seed to transplanting
In it, incubation time is shortened, improves pseudobulb differentiation rate, the fast-growth of pseudobulb is promoted, improves tissue-cultured seedling
Transplanting survival rate.
Detailed description of the invention
In order to keep the purpose of the present invention, technical scheme and beneficial effects clearer, the present invention provides following attached drawing and carries out
Illustrate:
Fig. 1 is the diameter test chart of one plant of bletilla pseudobulb；
The plant height test chart of Fig. 2 bletilla pseudobulb；
The plant height test chart of Fig. 3 bletilla pseudobulb；
Fig. 4 is a bletilla pseudobulb growing state after culture dish culture 60 days；
Fig. 5 is the growing state after transplanting 20 days.
Specific embodiment
Below in conjunction with attached drawing, a preferred embodiment of the present invention will be described in detail.
Following embodiment (is adopted in Chongqing City's drug plantation research institute experiment base using mature uncracked bletilla capsule
Ground), fruit pod store method, the fruit pod of micro- Huang is acquired in the 8-10 month, and ventilation is dried in the air 5-10 days indoors, becomes shallow to fruit pod epidermis
It when brown, is put into kraft envelope, is placed in 4 DEG C of fridge freshness retaining rooms, this method can save seed vitality and effectively convenient for week
The production in year.Culture medium high pressure sterilization 17min under the conditions of 121 DEG C, it is spare.
Seed fruit pod used in following embodiment is sterilized and is inoculated in the following manner: uncracked fruit pod flowing water is washed
Only, 1~3min of surface is washed with 75% alcohol painting, it is 0.1% that fruit pod, which is then placed in mass fraction, on superclean bench
15~20min is impregnated in mercuric chloride, is then used aseptic water washing 3-5 times, and aseptic filter paper blots surface moisture in case inoculation, is being inoculated with
The fruit pod sterilized is cut off into osculum with sterile scissors in disk, seed is dipped with aseptic inoculation ring and is uniformly coated in germination medium
On.
The screening of bletilla axenic germination culture medium
According to design is formulated shown in table 1, bletilla seed asepsis sprouting is observed, condition of culture are as follows: illumination
1500--2000Lx, 10-12h/ days；Temperature, 24--28 DEG C.It is as shown in table 2 that bletilla sprouting height of seedling, root hair situation are counted at 4 weeks:
Table 1: the formula design of bletilla aseptic culture medium
Table 2: bletilla seed germination and growth situation on different germination mediums
From table 2 it can be seen that in condition of culture, 1/2MS is good compared with MS culture effect under the same conditions, and several culture mediums
Comparison discovery, (4) culture medium prescription are sprouting starting, and seedling growth aspect is with the obvious advantage, so being more suitable for that bletilla is quickly sprouted
Hair needs, preferably germination medium formula are as follows: 1/2MS+NAA0.1mg/L+GA30.1mg/L+ white sugar 20g/L.
Bulb efficiently induces and the screening of strong seedling culture base
By the bulb of high 0.8~1.2cm be transferred to table 3 enumerate shown in carry out switching culture, condition of culture in culture medium
For pH5.8-6.0；Illumination 1500--2000Lx, 12-15h/ days；Temperature, 24--28 DEG C；To the bulb growth to each culture medium
Situation is observed and counts, and is recorded in table 4.
3 bulb of table efficiently induces and the design of strong seedling culture based formulas
4 bulb of table efficiently induces, strong seedling culture base screens situation
As seen from Table 4, A formula root growth is prosperous, affects the differentiation and growth of bulb；H culturing gene paclobutrazol concentration
It is higher, it is obvious to there is point evil, and bulb, plant strain growth are all suppressed；D, E, F be formulated, root growth is good, but bulb differentiation and
Growing way is poor, breaks up more very thin seedling；B, C, G are formulated, because of 6-BA, PP333Concentration and proportion more be suitable for, bulb
Differentiation and growing way are preferable, wherein with C formula, effect is most significant in terms of bulb induces differentiation, bulb growth, strong sprout, so being suitable for
Bletilla bulb efficiently induces, the formula of strong seedling culture is 1/2MS, NAA0.6mg/L, 6-BA0.8mg/L, PP3330.5mg/L is white
Sugared 40g/L, banana puree 25g/L, active carbon 0.1g/L.
Tissue-cultured seedling made of further table 4 is cultivated (switching 60 days) is transplanted, and is opened hardening 1 week, is transplanted to covering
On the ridge of fertile soil 3-5cm, and pay attention to shading and moisturizing, survival rate and growing way counted when transplanting 20 days, is recorded in shown in table 5:
Table 5: the survival rate statistics of transplanted seedling
" _ ", indicates that long potential difference, "+" indicate that growing way is preferable.
As can be seen from Table 5, the transplanted seedling growth potential and situation of taking root of C formula are preferable.
1 bletilla bulb rapid induction tissue culture method of embodiment
Step 1: fruit pod requirement, using the brown that shoals, mature bletilla fruit pod, the fruit pod of routine preservation is easy to infection miscellaneous bacteria
And rupture, it is unable to satisfy needed for bletilla tissue-cultured seedling whole year production, being encased using brown paper in refrigerator cold-storage is 4 DEG C of preservations；
Step 2: surface 1-3min is washed into the 70% alcohol painting of uncracked fruit pod, then by fruit on superclean bench
Pod, which is placed in the mercuric chloride that mass fraction is 0.1%, impregnates 15--20min, then with aseptic water washing 3--5 times, aseptic filter paper is blotted
Surface moisture is in case inoculation；On superclean bench, the fruit pod sterilized is cut off into osculum with sterile scissors, with aseptic inoculation ring
It dips seed to be uniformly coated on the culture medium of high-temperature sterilization, culture medium prescription 1/2MS+NAA0.1mg/L+GA30.1mg/L+
Agar powder 6.5-7.0g/L+ white sugar 20g/L, pH5.8-6.0；Condition of culture is that condition of culture is illumination 1500--2000Lx,
10-12h/ days；Temperature, is cultivated 4 weeks or so by 24--28 DEG C, obtains band 1-2 piece leaflet, the seedling of plant height 0.8-1.2cm；
Step 3: carrying out switching culture, culture medium prescription 1/2MS+NAA0.6mg/L+6-BA0.8mg/L+PP3330.5mg/L
+ white sugar 40g/L+ banana puree 25g/L+ active carbon 0.1g/L, pH5.8-6.0, are used after sterilized；The culture medium is bletilla pseudobulb
Induction, fast-growth, the strong seedling culture base taken root；Condition of culture: illumination 1500--2000Lx, 12-15h/ days；Temperature, 24--
28℃；On switching culture medium, pseudobulb induction and fast-growth observation: switching seedling passes through on pseudobulb efficient culture medium
It cultivates within 2-3 weeks, small pseudobulb has broken up, 4-5 weeks Shi Tongji, and bulb differentiation rate is 90% or more, and pseudobulb growth is fast
Speed；When by 8 weeks, it is 90% or more that pseudobulb diameter, which is greater than 0.4cm, plant height 6-10cm, and root is flourishing；Fig. 1 is one plant of bletilla
The diameter test chart of pseudobulb, its diameter reaches 9.41mm as can be seen from Figure 1, and Fig. 2, Fig. 3 are the plant height of two plants of bletilla pseudobulbs
Test chart, Fig. 4 are a bletilla pseudobulb growing state after culture dish culture 60 days.
Step 4: cultivating seedling is opened hardening 1 week from being moved on under normal temperature environment in culturing room, is cleaned by hardening, transplanting, field planting
Onto the case face for transplanting seedlings ground, root water is watered, and with the sunshade net sunshade of sunshade rate 40-60%, water 1 time within every 3-5 days, holding
Humidity.30 days or so, new root and young leaves were grown, and counted survival rate, and Fig. 5 is the growing state after transplanting 20 days.Use above method
Culture, respectively on March 25th, 2017, on April 22nd, 2017, on May 20th, 2017,22 sunrise transplantation of seedlings June in 2017, at
Motility rate statistics such as the following table 6:
Table 6: the transplanting survival rate of the tissue-cultured seedling of embodiment 1
As can be seen from Table 6, the young plant survival rate of each date transplanting is 85% or more, and fast growing；And May
Part, it is that transplanting survival rate is best on overburden soil with fertile soil 70%+ perlite 30%.
Bletilla bulb tissue culture method described in embodiment 1 is compared with existing method further, is obtained as shown in table 7
Conclusion:
The comparison of 7 existing method of table and 1 the method for embodiment
In table " --- " indicate without
It can further be reflected by table 7, this method simplifies the production link of bletilla pseudobulb tissue-cultured seedling, induces false squama
Stem promotes pseudobulb fast-growth and Rooting and hardening-off culture to complete in same step, is 90 days the time required to from seed to transplanting
It is interior, incubation time is shortened, pseudobulb differentiation rate is improved, promotes the fast-growth of pseudobulb, improves the shifting of tissue-cultured seedling
Plant survival rate.
Finally, it is stated that preferred embodiment above is only used to illustrate the technical scheme of the present invention and not to limit it, although logical
It crosses above preferred embodiment the present invention is described in detail, however, those skilled in the art should understand that, can be
Various changes are made to it in form and in details, without departing from claims of the present invention limited range.
Claims (6)
1. a kind of special culture media for inducing bletilla bulb, which is characterized in that the special culture media is sprouted by bletilla seed to be trained
Support base and bulb induction and strong seedling culture base composition, in which:
The formula of the bletilla seed germination medium are as follows: 1/2MS, NAA0.1mg/L, GA30.1mg/L, 6.5~7.0g/ of agar
L, white sugar 20g/L, pH5.8~6.0；
The formula of the bulb induction and strong seedling culture base are as follows: 1/2MS, NAA0.5~0.6mg/L, 6-BA0.6~0.8mg/L,
PP3330.4~0.5mg/L, white sugar 30-45g/L, 25~40g/L of banana puree, active carbon 0.1g/L, pH5.8~6.0.
2. a kind of special culture media for inducing bletilla bulb according to claim 1, which is characterized in that bulb induction and
The formula of strong seedling culture base are as follows: 1/2MS, NAA0.6mg/L, 6-BA0.8mg/L, PP3330.5mg/L, white sugar 40g/L, banana puree
25g/L, active carbon 0.1g/L, pH5.8-6.0.
3. a kind of tissue culture method for inducing bletilla bulb, which comprises the steps of:
1) surface 1- the sterilization of bletilla seed: is washed into the alcohol painting that uncracked bletilla seed mass fraction is 75%
Fruit pod, is then placed in 0.1% mercuric chloride 15~20min of immersion by 3min on superclean bench, then with aseptic water washing 3~5
Secondary, aseptic filter paper blots surface moisture in case inoculation；
2) under sterile operating condition, the bletilla seed of step 1) sterilization is inoculated into seed described in claim 1
It is cultivated in germination medium；
3) bulb described in claim 1 is forwarded to when the bletilla seed described in the step 2 sprouts 0.8~1.2cm of height of seedling to lure
Lead and strong seedling culture base in cultivate.
4. a kind of tissue culture method for inducing bletilla bulb according to claim 3, which is characterized in that the condition of culture of step 2
Are as follows: illumination 1500~2000Lx, 10~12h/ days；Temperature, 24~28 DEG C.
5. a kind of tissue culture method for inducing bletilla bulb according to claim 3, which is characterized in that the condition of culture of step 3)
Are as follows: illumination 1500~2000Lx, 12~15h/ days；Temperature, 24~28 DEG C.
6. a kind of tissue culture method for inducing bletilla bulb according to claim 3, which is characterized in that the bletilla seed is adopted
Collection and storage practice are as follows: the place that acquisition bletilla in 9~October is mature, full fruit pod is placed in shady and cool ventilation, spreading for cooling 2~3 weeks；To fruit
When pod epidermis becomes light brown, fruit pod is loaded in kraft paper bag and is stored in 4 DEG C of refrigerating chambers of refrigerator, it is ensured that bletilla tissue-cultured seedling
Used in whole year production.
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| CN108541593A (en) * | 2018-06-27 | 2018-09-18 | 芜湖东源新农村开发股份有限公司 | The method for tissue culture of white taro |
| CN109964797A (en) * | 2019-03-22 | 2019-07-05 | 浙江大学 | The method for promoting bletilla pseudobulb to be proliferated/expand |
| CN109964815A (en) * | 2019-03-26 | 2019-07-05 | 成都大学 | A kind of bletilla striata aseptic seedling rapid induction Multiple Buds and fast numerous method of taking root |
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