CN110396495A - Himalayan four-o'clock root callus, enrichment procedure and suspension cell propagation method - Google Patents
Himalayan four-o'clock root callus, enrichment procedure and suspension cell propagation method Download PDFInfo
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Abstract
The present invention relates to field of plant tissue culture technique, a kind of himalayan four-o'clock root callus, enrichment procedure and suspension cell propagation method are specifically disclosed.Himalayan four-o'clock root callus induction enrichment procedure of the invention is that after carrying out Fiber differentiation formation callus with blade of the induced medium to himalayan four-o'clock root aseptic seedling, the further Multiplying culture of this callus proliferated culture medium is obtained subcultured callus；Wherein, inducing with the hormone in proliferated culture medium is TDZ and NAA.This method inductivity is high, differentiation less, growth cycle it is short, resulting callus is soft frangible, is conducive to later use.It is that suspension culture is carried out to the himalayan four-o'clock root callus being prepared with the fluid nutrient medium comprising TDZ, NAA and caseinhydrolysate the present invention also provides a kind of himalayan four-o'clock root suspension cell propagation method.This method is easy to operate, cell proliferation rate is high, and growth cycle is short, at low cost, it can be achieved that industrialization whole year production.
Description
Technical field
The present invention relates to field of plant tissue culture technique.Specifically, being related to a kind of himalayan four-o'clock root callus group
It knits, enrichment procedure and suspension cell propagation method.
Background technique
Qinghai-Tibet (Qinghai-Tibet Plateau) is Largest In China, the highest plateau of world's height above sea level, referred to as
" roof of the world ", the earth " third pole ".With radiation is strong, sunshine is more, temperature is low, accumulated temperature is few, temperature is with height and latitude
Increase and reduce, day and night temperature it is big；Dry and wet is clearly demarcated, more night rain；Winter it is dry and cold it is very long, strong wind is more；Summer temperature it is cool it is rainy, hail is more
Etc. typical plateau climate feature.Under such environmental condition, only those generations and intense radiation, ice and snow, severe cold, strength
The adverse weather conditions such as wind, flowstone, barren are made to obtain special ecology-biology meeting market's demand plant in indomitable struggle,
It could survive, settle down and multiply.
Tibetan medicine and pharmacology is that Tibetan people combines the medicinal experience of tradition, Hindu medicine, Chinese medicine etc. in the long-term struggle with disease
The medicine systems with Qinghai-Tibet materia medica and national characters that traditional medicine is formed.Since Qinghai-Tibet Platean is unique certainly
Right ecological condition, the medicinal ingredient abundant contained in distinctive plateau plant become the material base of Tibetan medicine and pharmacology.
Himalayan four-o'clock root (Mirabilis himalaica) is Nyctaginaceae (Nyctaginaceae) Mirabilis
(Mirabilis) herbaceos perennial, Tibetan language transliteration " bar Zhu ", wild resource focuses primarily upon height above sea level 3000-3400m's
Tibet, the hillside thick grass of Northwest Sichuan and India part, waste rand or shrub forest area.
Himalayan four-o'clock root usage history in Tibetan medicine is long, in the Tibetan medicine and pharmacology masterpiece Four-Volume Medical Code and " Jingzhubencao "
It is on the books, it is most common medicinal material in China's Tibetan medicine and pharmacology ancient books and records.It is used as medicine with root, has warm kidney, kidney-nourishing nourishing, myogenic, benefit
Urine, row's stone and other effects, and with rhizoma polygonati (Polygonatum verticillatum), puncture vine (Tribulus terretris), west
Hiding rib celery (Pleurospermum hookeri), lucid asparagus (Asparaguscochinchinensis) are collectively known as passing
" five " of system Tibetan medicine mainly appear in the *** traditional medicine formula with tonic effect, are ten five tastes catechu ball of Tibetan medicine, Ba Sangmu
One of primary raw materials of dosage forms such as butter ball, 25-component Podophyllum emodi var chinense ball, Re Baluobu cream.
Although type plant resources abundant have been bred in Qinghai-Tibet Platean, the resource reserve for being faced with species is limited, kind
Group's renewal speed is slow, is difficult to restore after collection of resources, industry development brings resource exhaustion, ecological environment destruction etc. that can not hold in succession
Continuous a series of problems, such as utilizing.It is found according to on-site inspection in recent years, with the development of Tibetan medicine and pharmacology cause, since the excess of people is adopted
Collection, the wild resource of most common medicinal material himalayan four-o'clock root are constantly reduced, and wild resource is being petered out, and 2005
End of the year Tibet Science and Technology Department holds symposium, and the medicinal material is classified as level-one Tibetan medicine material in imminent danger.
Plant tissue and cell culture is removed not to be influenced by natural environment (such as weather, season, region, natural calamity) factor
Except, the yield and quality of metabolin is also more stable, can farmland saving, solve the problems, such as to treasure plant resources and lack and in imminent danger.
It explores new synthetic route and obtains new product, especially to endangered, slow growth, growth cycle length, growing environment
Badly, the plant resources production for cultivating medicinal plant and high economic value at high cost has more irreplaceable advantage, nothing
By all having particularly important application value from ecological benefits, economic benefit or social benefit.Currently, have more than 60% it is anti-
Cancer medicine and the drug of 75% treatment disease all derive from natural products.Plant Tissue Breeding is widely used to agricultural, forestry, doctor
The fields such as medicine, chemical industry produce nearly 1000 kinds of plant of secondary metabolite, and the active constituent more than 600 kinds is directly by plant
Cell culture provides.
Although artificial growth himalayan four-o'clock root has made great progress in recent years, due to himalayan four-o'clock root medicine
It is perennial root with position, growth cycle is long, is easily influenced by environmental conditions, it is difficult to meet Tibet's drug industry to happiness horse traction
The wilderness demand of refined four-o'clock.It would therefore be highly desirable to find a kind of more effective himalayan four-o'clock root modes of reproduction.
Summary of the invention
In order to solve the problems in the existing technology, high, growth that it is an object of the present invention to provide a kind of inductivities
Fastly, differentiation less, the stable himalayan four-o'clock root callus induction enrichment procedure of growth and thus method be prepared it is shallow
Yellow, translucent, soft frangible himalayan four-o'clock root callus.
It is a further object of the present invention to provide the fast himalayan four-o'clock root suspension cells of a kind of good dispersion, the speed of growth
Propagation method.
In order to achieve the object of the present invention, technical solution provided by the invention is as follows:
A kind of himalayan four-o'clock root callus induction enrichment procedure, comprising:
Fiber differentiation is carried out with blade of the induced medium to himalayan four-o'clock root aseptic seedling and forms callus, by institute
It states the further Multiplying culture of callus proliferated culture medium and obtains subcultured callus；
Wherein, the hormone in the induced medium and the proliferated culture medium is TDZ and NAA.
The present invention is had found by numerous studies, using TDZ and NAA as the hormone in induced medium, can be succeeded efficient
The callus primary of himalayan four-o'clock root is induced, and further using TDZ and NAA as the hormone in proliferated culture medium,
So that callus primary can both be grown with fast and stable, in turn avoids excessively breaking up, realize efficient, high-quality Himalaya
Four-o'clock callus (subcultured callus) preparation.
Preferably, the induced medium are as follows: MS+0.5-3.0mg/L TDZ+0.5-4.0mg/L NAA, more preferably
Are as follows: MS+1.5mg/L TDZ+2.0mg/L NAA.
The present invention uses MS for basic culture medium, cooperates with the NAA of the TDZ and 0.5-4.0mg/L of 0.5-3.0mg/L and carries out
The induction of himalayan four-o'clock root callus primary, enables to dedifferentiation effect preferable, and optimization formula is MS+
When 1.5mg/L TDZ+2.0mg/L NAA, higher inductivity (healing rate 96.67%) can be further obtained.
Preferably, the proliferated culture medium are as follows: MS+0.5-2.0mg/L TDZ+1.0-3.0mg/L NAA, more preferably
Are as follows: MS+1.0mg/L TDZ+2.0mg/L NAA；
The present invention uses MS for basic culture medium, cooperates with the NAA of the TDZ and 1.0-3.0mg/L of 0.5-2.0mg/L and carries out
The shoot proliferation of himalayan four-o'clock root callus primary, enables to that callus proliferation is fast, cell differentiation is few, and cell is raw
It is long to stablize, and when optimization formula is MS+1.0mg/L TDZ+2.0mg/L NAA, it can further obtain better cultivation effect.
As a kind of preferred embodiment of the invention, method of the present invention includes:
(1) himalayan four-o'clock root aseptic seedling is cultivated；
(2) it by the blade inoculation of the aseptic seedling into the induced medium, is lured under 25 ± 2 DEG C of dark conditions
Culture 30-40 days is led, the callus is obtained；
(3) callus is inoculated into the proliferated culture medium, proliferation training is carried out under 25 ± 2 DEG C of dark conditions
It supports 10-20 times, each 20-30 days, obtains subcultured callus.
Preferably, it carries out disinfection to the himalayan four-o'clock root seed and is used further to culture aseptic seedling after handling.Disinfection side
Known various methods can be used in formula, are such as carried out disinfection with the combination of+2% sodium hypochlorite of 75% alcohol, the present invention does not limit especially
It is fixed.
Preferably, the cultural method of the aseptic seedling are as follows: with MS culture medium in 25 ± 2 DEG C, intensity of illumination 2500-
To himalayan four-o'clock root seed culture 20-30 days under the conditions of 3500lx, daily light application time 16h, dark 8h.
It is highly preferred that Himalaya purple jasmine under the conditions of 25 DEG C, intensity of illumination 3000lx, 16 hour/day of light application time
Jasmine seed culture 25 days.This mode is more advantageous to himalayan four-o'clock root Aseptic Seedling Growth.
Preferably, the seedling leaves of aseptic seedling is taken to be inoculated with.
Preferably, induction of callus carries out 35 days under 25 DEG C of dark conditions, this mode can be more advantageous to happiness horse
Refined four-o'clock explant is drawn to generate callus.
Preferably, callus proliferation culture carries out 15 times under 25 DEG C of dark conditions, and 25 days every time, this mode can more have
Conducive to forming light yellow, translucent, soft frangible callus.
Preferably, the induced medium and the proliferated culture medium also include the sucrose of 25-30g/L, 6.5-7.0g/L
Agar.Water in the induced medium and the proliferated culture medium is deionized water.
The present invention provides energy by callus of sucrose, using agar as support.
Preferably, the induced medium and the pH value of the proliferated culture medium are 5.8-6.0, more preferably 5.8.
The present invention also provides a kind of himalayan four-o'clock root callus prepared by the above method.
The himalayan four-o'clock root callus be it is light yellow, translucent, soft frangible, conducive to stable subculture is carried out
Culture is more advantageous to carry out suspension cell culture and expand and give birth to compared to the conventional callus for only carrying out induction primary and obtaining
It produces.
The present invention separately provides a kind of himalayan four-o'clock root suspension cell propagation method, comprising: with fluid nutrient medium to upper
It states the himalayan four-o'clock root callus being prepared and carries out suspension culture, the fluid nutrient medium includes TDZ, NAA and water
Solve casein.
Preferably, it is described suspend culture number be 5-10 times, each 15-30 days, to obtain well-grown, good dispersion
And the suspension cell that the speed of growth is fast.
It is highly preferred that the number of the culture that suspends is 10 times, every time 20 days.
Preferably, the suspension cell breeding carries out on shaking table, shaking speed 80-120r/min；And/or it is described
Suspension cell breeding carries out under 25 ± 2 DEG C of dark conditions.
Preferably, the inoculum concentration of himalayan four-o'clock root callus is 60-80g/L in the fluid nutrient medium.
Preferably, the fluid nutrient medium is MS+0.5-2.0mg/L TDZ+1.5-3.0mg/L NAA+100-300mg/L
Caseinhydrolysate, preferably are as follows: MS+1.0mg/L TDZ+2.0mg/L NAA+200mg/L caseinhydrolysate；
The present invention uses MS for basic culture solution, cooperates with the NAA of the TDZ and 1.5-3.0mg/L of 0.5-2.0mg/L and carries out
Suspension cell breeding, enables to that suspension cell growth is stable, scattered, proliferation is fast, and optimization formula is MS+1.0mg/L
When TDZ+2.0mg/L NAA+200mg/L caseinhydrolysate, reproductive effect can be further promoted.Wherein, a certain proportion of hydrolysis
Casein has facilitation to cell suspension cultures.
Preferably, the fluid nutrient medium also includes the sucrose of 25-30g/L；And/or the pH value of the fluid nutrient medium
For 5.8-6.0, preferably 5.8.
Preferably, the water in the fluid nutrient medium is deionized water.
Beneficial effects of the present invention at least that:
Method of the invention can be quickly obtained a large amount of Tibet himalayan four-o'clock root well-grown, hereditary capacity is stablized
Callus, inductivity is high, differentiation less, growth cycle it is short, at low cost.
The himalayan four-o'clock root callus that the present invention obtains is in light yellow, translucent, soft frangible state, is conducive to
Suspension cell screening and squamous subculture are carried out, is the desirable feedstock as the breeding of subsequent suspension cell.
Suspension cell propagation method of the invention, easy to operate, cell proliferation rate is high, growth cycle is short, at low cost, can be with
Tibet himalayan four-o'clock root suspension cell pharmaceutical raw material is provided quantity-unlimitingly, realizes the industrialization anniversary of himalayan four-o'clock root
Production, had not only realized the development and utilization of resource, but also advantageously reduce the dependence to wild himalayan four-o'clock root resource,
It can solve the problems, such as China's Tibetan medicine and pharmacology industry development, there is most important theories meaning and wide market application prospect.
Detailed description of the invention
Fig. 1 is himalayan four-o'clock root plant of the present invention；
Fig. 2 is part callus obtained in 1 step of the embodiment of the present invention (2)；
Fig. 3 is part subcultured callus obtained in 1 step of the embodiment of the present invention (3)；
Fig. 4 is suspension cell obtained in 1 step of the embodiment of the present invention (4).
Specific embodiment
The preferred embodiment of the present invention is described in detail below in conjunction with embodiment.It will be appreciated that following real
Providing merely to play the purpose of explanation for example is applied, is not used to limit the scope of the present invention.The skill of this field
Art personnel without departing from the spirit and purpose of the present invention, can carry out various modifications and replace to the present invention.
Experimental method used in following embodiments is conventional method unless otherwise specified.Institute in following embodiments
Material, reagent etc., are commercially available unless otherwise specified.
Material therefor in the present invention are as follows: MS culture medium (PhytoTechnology Laboratories company, the U.S.)；6-
BA (6-benzyladenine), NAA (methyl α-naphthyl acetate), TDZ (Thidiazuron), 2,4-D (2,4 dichlorophenoxyacetic acid), caseinhydrolysate,
Coconut palm cream (Beijing Baeyer enlightening Bioisystech Co., Ltd).
Embodiment 1
(1) culture of aseptic seedling
The seed for taking himalayan four-o'clock root (plant forms are referring to Fig. 1) maturation, according to+2% sodium hypochlorite of 75% alcohol
Combined conventional disinfection method carries out disinfection to it processing, is seeded on MS culture medium, cultivates aseptic seedling.Condition of culture: illumination
3000lx, daily light application time 16h, dark 8h, 25 DEG C of set temperature, incubation time 25 days.
(2) induction of callus
The blade (seedling leaves) of the aseptic seedling of step (1) culture is cut into 0.5cm × 0.5cm, accesses induced medium
Middle carry out induction of callus, obtains callus primary.Culture medium prescription are as follows: MS+1.5mg/L TDZ+2.0mg/L
NAA.Energy matter: sucrose 30g/L；Support: agar 6.5g/L；PH value: pH5.8；Solvent: in the composition of induced medium
Water is deionized water；Condition of culture: it is dark, 25 DEG C of set temperature；Incubation time: 35 days.
I.e. visible blade explant incision has thickening phenomenon after explant is inoculated with 10 days, visible yellow green occurs within 35 days
Finer and close graininess callus, referring to fig. 2.
(3) squamous subculture of callus
It takes the callus (callus primary) induced in step (2) to carry out squamous subculture and obtains subcultured callus.
Proliferation culture medium formula are as follows: MS+1.0mg/L TDZ+2.0mg/L NAA.Energy matter: sucrose 30g/L；Support: agar
6.5g/L；PH value: pH5.8；Solvent: water is deionized water in the composition of subculture medium；Condition of culture: dark, temperature 25
DEG C, it 25 days, recycles 15 times.Obtained subcultured callus, referring to Fig. 3.
(4) suspension cell culture
It will repeatedly be suspended in subcultured callus 6g access fluid nutrient medium 100mL loose after step (3) proliferation
Squamous subculture obtains himalayan four-o'clock root suspension cell.Culture medium prescription are as follows: MS+1.0mg/L TDZ+2.0mg/L NAA+
200mg/L caseinhydrolysate.Energy matter: sucrose 30g/L；PH value: pH5.8；Solvent: water in the composition of suspension cell culture
For deionized water；Condition of culture: dark, shaking speed 80r/min, 20 days, is recycled 10 times by 25 DEG C of temperature.It is obtained outstanding
Floating cell, referring to fig. 4.
Embodiment 2
Callus and suspension cell culture are carried out to himalayan four-o'clock root in the same manner as example 1, it is different
:
The condition of culture of aseptic seedling is illumination 2500lx, and incubation time is 30 days.
Callus inducing medium formula are as follows: MS+3.0mg/L TDZ+4.0mg/L NAA, energy matter: sucrose 25g/
L；Support: agar 7.0g/L；Incubation time: 30 days.
Subcultured callus proliferation culture medium formula are as follows: MS+0.5mg/L TDZ+3.0mg/L NAA, energy matter: sucrose
25g/L；Support: agar 7.0g/L；Incubation time: it 20 days, recycles 15 times.
Suspension cell culture based formulas are as follows: MS+2.0mg/L TDZ+3.0mg/L NAA+300mg/L caseinhydrolysate, energy
Quantity of material: sucrose 25g/L；Condition of culture: incubation time: shaking speed 120r/min 15 days, is recycled 10 times.
Embodiment 3
Callus and suspension cell culture are carried out to himalayan four-o'clock root in the same manner as example 1, it is different
:
The condition of culture of aseptic seedling is illumination 3500lx, and incubation time is 20 days.
Callus inducing medium formula are as follows: MS+0.5mg/L TDZ+2.0mg/L NAA, incubation time: 40 days.
Subcultured callus proliferation culture medium formula are as follows: MS+2.0mg/L TDZ+3.0mg/L NAA, incubation time: 30
It, recycles 20 times.
Suspension cell culture based formulas are as follows: MS+0.5mg/L TDZ+1.5mg/L NAA+100mg/L caseinhydrolysate, training
It supports the time: 30 days, recycling 5 times.
Comparative example 1
The himalayan four-o'clock root aseptic seedling being prepared in embodiment 1 is cured in the same manner as example 1
Injured tissue induction, obtains callus primary, unlike: callus inducing medium formula are as follows: MS+3.5mg/L TDZ+
4.5mg/L NAA。
Comparative example 2
The himalayan four-o'clock root aseptic seedling being prepared in embodiment 1 is cured in the same manner as example 1
Injured tissue induction, obtains callus primary, unlike: callus inducing medium formula are as follows: MS+1.5mg/L 6-BA
+2.0mg/L NAA。
Comparative example 3
The himalayan four-o'clock root aseptic seedling being prepared in embodiment 1 is cured in the same manner as example 1
Injured tissue induction, obtains callus primary, unlike: callus inducing medium formula are as follows: MS+1.5mg/L TDZ+
2.0mg/L 2,4-D。
Comparative example 4
In the same manner as example 1 to the himalayan four-o'clock root callus group being prepared in 1 step of embodiment (2)
It knits (callus primary) and carries out squamous subculture, unlike: proliferation culture medium formula are as follows: MS+2.5mg/L TDZ+3.5mg/L
NAA。
Comparative example 5
In the same manner as example 1 to the himalayan four-o'clock root callus group being prepared in 1 step of embodiment (2)
It knits (callus primary) and carries out squamous subculture, unlike: proliferation culture medium formula are as follows: MS+1.0mg/L 6-BA+2.0mg/
L NAA。
Comparative example 6
In the same manner as example 1 to the himalayan four-o'clock root callus group being prepared in 1 step of embodiment (2)
It knits (callus primary) and carries out squamous subculture, unlike: proliferation culture medium formula are as follows: MS+1.0mg/L TDZ+2.0mg/L
2,4-D。
Comparative example 7
Using the himalayan four-o'clock root subcultured callus being prepared in 1 step of embodiment (3) as raw material, using with reality
It applies the identical mode of example 1 and carries out suspension cell culture, unlike: suspension cell culture based formulas are as follows: MS+2.5mg/L TDZ+
3.5mg/L NAA+350mg/L caseinhydrolysate.
Comparative example 8
Using the himalayan four-o'clock root subcultured callus being prepared in 1 step of embodiment (3) as raw material, using with reality
It applies the identical mode of example 1 and carries out suspension cell culture, unlike: suspension cell culture based formulas are as follows: MS+1.0mg/L TDZ+
2.0mg/L NAA+350mg/L caseinhydrolysate.
Comparative example 9
Using the himalayan four-o'clock root subcultured callus being prepared in 1 step of embodiment (3) as raw material, using with reality
It applies the identical mode of example 1 and carries out suspension cell culture, unlike: suspension cell culture based formulas are as follows: MS+1.0mg/L TDZ+
2.0mg/L NAA+50mg/L caseinhydrolysate.
Comparative example 10
Using the himalayan four-o'clock root subcultured callus being prepared in 1 step of embodiment (3) as raw material, using with reality
It applies the identical mode of example 1 and carries out suspension cell culture, unlike: suspension cell culture based formulas are as follows: MS+2.5mg/L TDZ+
2.0mg/L NAA+200mg/L caseinhydrolysate.
Comparative example 11
Using the himalayan four-o'clock root subcultured callus being prepared in 1 step of embodiment (3) as raw material, using with reality
It applies the identical mode of example 1 and carries out suspension cell culture, unlike: suspension cell culture based formulas are as follows: MS+1.0mg/L TDZ+
3.5mg/L NAA+200mg/L caseinhydrolysate.
Comparative example 12
Using the himalayan four-o'clock root subcultured callus being prepared in 1 step of embodiment (3) as raw material, using with reality
It applies the identical mode of example 1 and carries out suspension cell culture, unlike: suspension cell culture based formulas are as follows: suspension cell culture base
Formula are as follows: MS+1.0mg/L TDZ+2.0mg/L NAA+200mg/L coconut palm cream.
Experimental example 1
Observation statistics is carried out to callus primary obtained in 1-3 of the embodiment of the present invention, comparative example 1-3, the results are shown in Table
1。
Table 1
| It is inoculated with number | Healing rate % | Melting brown rate % | |
| Embodiment 1 | 30 | 96.67 | 6.67 |
| Embodiment 2 | 30 | 93.30 | 6.67 |
| Embodiment 3 | 30 | 83.30 | 6.67 |
| Comparative example 1 | 30 | 56.67 | 16.67 |
| Comparative example 2 | 30 | 26.70 | 46.67 |
| Comparative example 3 | 30 | 20.00 | 56.67 |
In table 1, healing rate (%)=induced synthesis dense callus block number/inoculation number × 100%；Melting brown rate (%)
Callus block number/inoculation block number × 100% of=browning.
As can be seen from the data in table 1: the healing rate of embodiment 1-3 is significantly larger than comparative example 1-3；Meanwhile comparative example 2,3 is brown
Rate is much higher than embodiment 1-3.
It follows that TDZ and NAA combination is to the inducing effect of callus primary better than 6-BA and NAA, TDZ and 2,4-D
Combination, and TDZ, NAA dosage it is excessively high when will be so that healing rate reduces, melting brown rate increases, and can not obtain ideal induction effect
Rate.
Himalayan four-o'clock root blade (explant) of the invention is visible blade explant incision after being inoculated with 10-20 days
There is thickening phenomenon, occurs the finer and close graininess callus of visible yellow green after 30-40 days, and healing rate is high, melting brown rate
It is low.
Experimental example 2
Observation statistics is carried out to subcultured callus obtained in 1-3 of the embodiment of the present invention, comparative example 4-6, the results are shown in Table
2。
Table 2
In table 2, differentiation rate (%)=differentiation block number/inoculation number × 100%；Melting brown rate (%)=browning subculture callus
Organize block number/inoculation block number × 100%.
From the data of table 2: the differentiation rate of embodiment 1-3 subcultured callus is far below comparative example 4-6；Meanwhile it is right
The melting brown rate of ratio 4-6 is much higher than embodiment 1-3.
It follows that hormone required for himalayan four-o'clock root callus proliferation selects the combination of TDZ and NAA to want excellent
In 6-BA and NAA, TDZ and 2, when the dosage of the combination of 4-D, and TDZ, NAA are excessively high will so that differentiation rate, melting brown rate all increase,
Ideal proliferation efficiency can not be obtained.
After multiple subculture, callus is constantly proliferated himalayan four-o'clock root callus of the invention, last shape
At light yellow, translucent, soft frangible callus (subcultured callus), and differentiation rate, melting brown rate are low.
Experimental example 3
Observation statistics is carried out to suspension cell obtained in 1-3 of the embodiment of the present invention, comparative example 7-12, the results are shown in Table 3:
Table 3
In table 3, fresh weight × 100% before proliferation rate (%)=(fresh weight before fresh weight-proliferation after proliferation)/proliferation, fresh weight test
Method is that cell suspending liquid is filtered by vacuum to exclude fluid nutrient medium, with distilled water flushing 3 times, claims its fresh matter after filtering draining
Amount.
From the data of table 3: embodiment 1-3 suspension cell proliferation rate is much higher than comparative example 7-12.TDZ's and/or NAA
(referring to comparative example 7,10,11), suspension cell proliferation slowly, is unsuitable for the training of himalayan four-o'clock root suspension cell when dosage is excessively high
It supports.It will all make suspension cell proliferation slow (referring to comparative example 8-9) when caseinhydrolysate dosage is excessively high, too low.Caseinhydrolysate
It is newborn (referring to comparative example 12) better than coconut palm to the growth and breeding effect of himalayan four-o'clock root suspension cell.
The himalayan four-o'clock root suspension cell good dispersion that the present invention obtains, proliferation rate are high.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, fall within the scope of the claimed invention without departing from theon the basis of the spirit of the present invention.
Claims (10)
1. a kind of himalayan four-o'clock root callus induction enrichment procedure characterized by comprising
Fiber differentiation is carried out with blade of the induced medium to himalayan four-o'clock root aseptic seedling and forms callus, described will be cured
The further Multiplying culture of injured tissue proliferated culture medium obtains subcultured callus；
Wherein, the hormone in the induced medium and the proliferated culture medium is TDZ and NAA.
2. the method according to claim 1, wherein the induced medium are as follows: MS+0.5-3.0mg/L TDZ+
0.5-4.0mg/L NAA。
3. method according to claim 1 or 2, which is characterized in that the proliferated culture medium are as follows: MS+0.5-2.0mg/L
TDZ+1.0-3.0mg/L NAA。
4. method according to claim 1-3 characterized by comprising
(1) himalayan four-o'clock root aseptic seedling is cultivated；
(2) by the blade inoculation of the aseptic seedling into the induced medium, induction training is carried out under 25 ± 2 DEG C of dark conditions
It supports 30-40 days, obtains the callus；
(3) callus is inoculated into the proliferated culture medium, Multiplying culture is carried out under 25 ± 2 DEG C of dark conditions
10-20 times, each 20-30 days, obtain subcultured callus.
5. method according to claim 1-4, which is characterized in that the cultural method of the aseptic seedling are as follows: with MS
Culture medium is under the conditions of 25 ± 2 DEG C, intensity of illumination 2500-3500lx, 16 hour/day of light application time to himalayan four-o'clock root kind
Son culture 20-30 days.
6. method according to claim 1-5, which is characterized in that the induced medium and the Multiplying culture
Base also includes the agar of the sucrose of 25-30g/L, 6.5-7.0g/L；And/or the induced medium and the proliferated culture medium
PH value be 5.8-6.0.
7. a kind of himalayan four-o'clock root callus, which is characterized in that prepared by method described in any one of claims 1-6
It obtains.
8. a kind of himalayan four-o'clock root suspension cell propagation method characterized by comprising wanted with fluid nutrient medium to right
7 himalayan four-o'clock root callus is asked to carry out suspension culture, the fluid nutrient medium includes TDZ, NAA and hydrolysis junket egg
It is white；It is preferred that the number of the culture that suspends is 5-10 times, each 15-30 days.
9. according to the method described in claim 8, it is characterized in that, the fluid nutrient medium is MS+0.5-2.0mg/L TDZ+
1.5-3.0mg/L NAA+100-300mg/L caseinhydrolysate.
10. method according to claim 8 or claim 9, which is characterized in that the fluid nutrient medium also includes the sugarcane of 25-30g/L
Sugar；And/or the pH value of the fluid nutrient medium is 5.8-6.0.
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