CN106718879B - The inducing culture and abductive approach of a kind of Radix zanthoxyli callus and application - Google Patents
The inducing culture and abductive approach of a kind of Radix zanthoxyli callus and application Download PDFInfo
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- CN106718879B CN106718879B CN201611044472.2A CN201611044472A CN106718879B CN 106718879 B CN106718879 B CN 106718879B CN 201611044472 A CN201611044472 A CN 201611044472A CN 106718879 B CN106718879 B CN 106718879B
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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
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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 inducing culture of Radix zanthoxyli callus and abductive approach and applications；The present invention is using stem section of the Radix zanthoxyli without offspring as explant, and evoked callus is formed on the MS culture mediums matched containing optimum hormone；Obtained callus can provide material for Radix zanthoxyli cell fusion or genetic transformation research, establishing techniques platform lays the first stone for the improvement of germ plasm resource, and subsequent tissue cultures are carried out using callus of the present invention, the tissue culturing system of foundation, improves breeding coefficient（It is existing to realize that the growth coefficient of the method for proliferation is 2.4~3.6 by the induction of Multiple Buds, and the growth coefficient of the method for the present invention can reach 5.0 or more）, lay the foundation for a large amount of plantation Radix zanthoxylis.
Description
Technical field
The present invention relates to technical field of tissue culture, more particularly, to a kind of Fiber differentiation of Radix zanthoxyli callus
Base and abductive approach and application.
Background technology
Radix zanthoxyli（Zanthoxylum nitidum (Roxb.) DC.）For Rutaceae xanthoxylum, have very high
Medical value, dry root are used as medicine, and are southern region of China common Chinese medicine, 1977 start to be incorporated into《Chinese Pharmacopoeia》.Two sides
Needle set has the effect of promoting qi circulation and relieving pain, promoting blood circulation and removing blood stasis, dispelling wind and removing obstruction in the meridians.Nitidine Chloride therein can be with anti-liver cancer and anti-and breast cancer；
The raw material of Radix zanthoxyli or toothpaste industry.Radix zanthoxyli is distributed in south China each province, is born in the bushes of hill hillside fields.Radix zanthoxyli
Although distribution is wide, resource is not enriched.Since Radix zanthoxyli dosage is huge, wild resource is almost exhausted, in the market two sides
The adulterant of medicine administered by injection material is therefore also more.Restore the resource of Radix zanthoxyli firstly the need of seedling.It can be sprouted by seed, cutting propagation
Seedling is obtained with tissue cultures.In addition Radix zanthoxyli germ plasm resource more lacks, with cell-fusion techniques and gene transformation technology
Continuous development, the expansion that can greatly promote germ plasm resource is improved plant on a molecular scale, to plant in molecular level
It improves and needs first to handle plant, usually induced synthesis callus, there are no the callus of Radix zanthoxyli in the prior art
The cultural method of tissue.
Invention content
The technical problem to be solved by the present invention is to overcome drawbacks described above of the existing technology, a kind of Radix zanthoxyli is provided and is cured
The inducing culture of injured tissue.
Second object of the present invention is to provide a kind of abductive approach of Radix zanthoxyli callus.
Third object of the present invention is to provide the applications of above-mentioned inducing culture or the abductive approach of callus.
The purpose of the present invention is what is be achieved by the following technical programs：
Application of the 6-nonylaminopurine in Radix zanthoxyli callus induction, the 6-nonylaminopurine is in culture medium
In a concentration of 0.2~6 mg/L.
Inventor, which studies, finds that 6-nonylaminopurine is added in Radix zanthoxyli（Also known as kinetin, abbreviation KT）It can induce out
Radix zanthoxyli callus carries out tissue cultures or the material of cell fusion and genetic transformation using callus, is Radix zanthoxyli
The expansion of germ plasm resource provides basis.
Preferably, a concentration of 1~4 mg/L of the 6-nonylaminopurine in the medium.
The present invention also provides a kind of Radix zanthoxyli callus inducing medium, the callus inducing medium is MS+
1.0~4.0 mg/LNAA of mg/L KT+1.0~2.0.
It is highly preferred that the callus inducing medium is MS+4.0 mg/L KT+2.0 mg/LNAA.
It is using stem section of the Radix zanthoxyli without offspring as explant the present invention also provides a kind of method of Radix zanthoxyli callus induction
Body carries out the induction of callus；The callus inducing medium is KT+1.0~2.0 the mg/L of MS+1.0~4.0
mg/LNAA。
Preferably, induction of callus condition is：23~27 DEG C of temperature, relative humidity 40%~60%, intensity of illumination
For 32.5~34.5 μm of olm-2·s-1, the photoperiod is that illumination and interlunation are fifty-fifty.
Preferably, stem section of the Radix zanthoxyli without offspring is to take the tender stem segments of the Radix zanthoxyli with axillary bud, after disinfection, be inoculated in
In the mg/L KT+0.2 mg/L of mg/L~0.4 of MS+1.0 mg/L~2.0 NAA culture grow up to 3~4cm without offspring, then cut
Take the stem section of no offspring.
Compared with prior art, the invention has the advantages that：
The present invention is induced using stem section of the Radix zanthoxyli without offspring as explant on the MS culture mediums matched containing optimum hormone
Callus is formed；Obtained callus can provide material for Radix zanthoxyli cell fusion or genetic transformation research, build
Vertical technology platform, lays the first stone for the improvement of seed resource, and subsequent tissue cultures are carried out using callus of the present invention,
The tissue culturing system of foundation, improves breeding coefficient（The existing proliferation that the method for proliferation is realized by the induction of Multiple Buds
Coefficient is 2.4~3.6, and the growth coefficient of the method for the present invention can reach 5.0）, lay the foundation for a large amount of plantation Radix zanthoxylis.
Specific implementation mode
The content further illustrated the present invention with reference to specific embodiment, but should not be construed as limiting the invention.
Without departing from the spirit and substance of the case in the present invention, to simple modifications or substitutions made by the method for the present invention, step or condition,
It all belongs to the scope of the present invention；Unless otherwise specified, technological means used in embodiment is well known to those skilled in the art
Conventional means.
Vegetable material：Radix zanthoxyli is without offspring stem section.Radix zanthoxyli tender stem segments with axillary bud pick up from Traditional Chinese Medicine University Of Guangzhou's medicine
With botanical garden, impregnated 30 seconds with 70% ethyl alcohol, then use 0.1%HgCl2Impregnate 8 minutes, with 0.5%NaClO impregnate 10 minutes, finally
With aseptic water washing 3 times, it is inoculated in MS+2.0 mgL-1KT+0.4 mgL-1It is cultivated in NAA, starts to give birth to after 12 days
It is long, about 12 Zhou Houke grow up to 3~4 cm without offspring.
It is basic culture medium, 30 gL of addition sucrose with MS culture mediums-1, 8.0 gL of carragheen-1, the works of pH5.8~6.0
For basic culture medium.Plant growth substance includes α-naphthylacetic acid（α-naphthalene acetic acid, NAA）, indoles fourth
Acid（Indole-3-butyric acid, IBA）, 6-nonylaminopurine（Kinetin, KT）.
Callus induction rate：Respectively in stem section callus Fiber differentiation the 10th, 20,30 day, callus growth feelings are observed
Condition counts inductivity（Inductivity=(going out more explant number/inoculation explant sum) × 100%）.
Phenylacetic acid：Callus differentiation culture 4 weeks or 8 weeks, counts phenylacetic acid（Differentiation rate=(point
Dissolve the callus total block data of callus block number/inoculation of bud) × 100%）.
Bud growth coefficient：After bud differentiation culture 8 weeks, it is forwarded in proliferated culture medium and cultivates.4 weeks or 8 weeks statistics buds of culture
Growth coefficient (bud growth coefficient=whole proliferation bud number/inoculation bud number).
Rooting rate：Culture of rootage 8 weeks counts rooting rate（Rooting rate=(the bud number for the bud number/inoculation taken root) × 100%）.
Embodiment 1
One, callus induction
The stem of no offspring is aseptically cut into the segment of about 6 mm, lies in a horizontal plane in callus inducing medium
Surface（The composition of callus inducing medium is specifically shown in Table 1）；Every bottle is inoculated with 3 sections；Respectively 25 bottles of the inoculation of each culture medium.Training
The condition of supporting：Temperature（25±2）DEG C, relative humidity 40%~60%, intensity of illumination is（33.6±0.7）μmol·m-2·s-1, light week
Phase is that illumination and interlunation are fifty-fifty, a month subculture 1 time.
The inducing culture that Radix zanthoxyli stem section is constituted in three kinds of different plant growth substance combinations（A1, A2, A3 culture medium by
Plant growth substance composition in basal medium and table 1, the content of wherein basal medium are identical）In can generate callus group
It knits, illustrates that the KT and NAA of debita spissitudo can induce generation callus.Compare the effect that three kinds of culture mediums generate callus
Fruit, the inductivity highest in A3 culture mediums, reaches 74.84%；Followed by A1 culture mediums, inductivity 66.84%；Worst is
A2 culture mediums, inductivity only have 58.76%.The quality of the callus of generation is also different, the callus group generated on A3 and A1
The color knitted is faint yellow, graininess, quality is loose and moistens；And the color of the callus generated on A2 is yellowish
Color, bulk, quality are stiff；Therefore the callus that generates on A3 and A1 culture mediums as callus relatively suitable for breaking up
Material, due to the inductivity highest on A3 culture mediums, so the callus for choosing the induction of A3 culture mediums carries out Analytical Chemical Experiment.
Two, the differentiation of callus
By subculture 1 time, the callus lines induced in A3 culture mediums are cut into the mm sizes of 5 mm × 5, are inoculated in differentiation training
It supports in base（Table 2, culture medium B1, B2, B3 be made of the plant growth substance of basal medium and table 2, wherein basal medium
Content is identical）, every bottle is inoculated with 4 pieces, respectively 50 bottles of the inoculation of each culture medium.After culture 1 month, it is cured what is generated on culture medium B3
Injured tissue is then transferred in another 4 kinds of differential mediums and cultivates（Table 3）, each 8 pieces of culture dish inoculation, the respectively inoculation 12 of each culture medium
A culture dish.Condition of culture：Temperature（25±2）DEG C, relative humidity 40%~60%, intensity of illumination is（33.6±0.7）μmol·
m-2·s-1, the photoperiod is that illumination and interlunation are fifty-fifty.
It the results are shown in Table 2.There is no the differentiation of bud in culture medium B1.And budding can be broken up on culture medium B2 and B3, but
Differentiation rate is low, and only 4.50% and 12.50%, the bud average of every piece of callus is 1.00 and 1.30.Do not have on culture medium B1
The reason of having differentiation budding may be that the concentration of KT is too low.From the results shown in Table 2, in three kinds of culture mediums, contain 4 mg/L
The B3 culture mediums of KT+0.2 mg/L IBA have certain effect to callus differentiation, but effect is ideal not enough.In order into
One step improves the differentiation rate of bud, the callus without bud of differentiation culture will be carried out in B3, and was transferred to other four kinds differentiation
Continue the differentiation culture of bud in culture medium（Table 3, B3, B4, B5, B6 by basal medium and table 3 plant growth substance group
At the content of wherein basal medium is identical）.
As can be seen from Table 3, the culture medium of four kinds of combinations containing plant growth substance can promote the differentiation of bud,
The differentiation effect of callus in culture medium B4 is preferable, inductivity 36.46%, is significantly higher than in other three culture mediums
In differentiation rate, and the average bud number of every piece of callus reaches 3.30.Callus bud in culture medium B3, B5 and B6
Differentiation rate is respectively 20.83%, 16.67% and 23.96%, does not have significant difference between them.In four kinds of culture mediums, the KT of B4
Content highest, the bud that differentiates is smaller, color is partially yellow, blade is tiny, illustrates the second-rate of bud.Therefore table 3 the result shows that,
The KT of high concentration is advantageous to the differentiation of bud, but the concentration of KT is too high, can make the degradation of bud.
The interpretation of result of consolidated statement 2 and table 3, bud differentiation culture in, in addition to the concentration of KT cannot it is too low other than, auxin
Concentration also has an impact the differentiation rate of bud, and in the identical processing of KT concentration, auxin concentration is high, then has inductivity is high to become
Gesture.In addition, inductivity of the inductivity of the B3 culture mediums of table 3 higher than the B3 culture mediums in table 2, reason may be table 3
The incubation time of callus is longer.
Three, the proliferation of bud
Band bud callus from differential medium B4 is transferred in bud proliferated culture medium（Table 4）, each culture dish
6 pieces of callus, 1~2 bud of every piece of callus band are inoculated with, each culture medium is respectively inoculated with 8 culture dishes.4 weeks subsequent generations 1
It is secondary, it is inoculated into culture bottle, the callus of every bottle of inoculation 3 pieces of bands, 1~2 bud, respectively 16 bottles of the inoculation of each culture medium.Cultivate item
Part：Temperature（25±2）DEG C, relative humidity 40%~60%, intensity of illumination is（33.6±0.7）μmol·m-2·s-1, the photoperiod is
Illumination is fifty-fifty with interlunation.
The differentiation rate of bud is apparently higher than other culture mediums in B4 culture mediums, but bud is second-rate, the possible reason is B4
The KT excessive concentrations of culture medium.In order to obtain the bud of high inductivity and good quality of getting back, by the bud weight on B4 culture mediums
It is newly transferred in B3, B5 and B6 culture medium of low KT concentration and carries out Multiplying culture.It the results are shown in Table 4.From table 4, it can be seen that culture 4
Week, most preferably B6 culture mediums, bud growth coefficient were 3.33 respectively to bud cultivation effect with after 8 weeks（Culture 4 weeks）With 5.00（Training
It supports 8 weeks）.Followed by B3 culture mediums, after cultivating 4 weeks and 8 weeks, bud growth coefficient is 2.66 and 3.50 respectively.Growth coefficient is higher
The KT concentration of the two culture mediums be all 4.0 mgL-1, their the bud speed of growth is slower, and bud is slightly shorter, but the color of bud compared with
It is green, illustrate that the quality of bud is good.Cultivation effect on B5 medium is worst, its a concentration of 2.0 mgL of KT-1.So
In Multiplying culture, the too low proliferation for being unfavorable for bud of the concentration of KT.
Comparing the plant growth substance composition in B5 and B6 culture mediums, the concentration proportion of their KT and NAA are all 5, but
The cultivation effect of the two bud is different, this explanation is identical with auxin ratio when the basic element of cell division, and their absolute concentration is not
Meanwhile the differentiation capability of bud is also variant, that is to say, that the differentiation capability of bud is not only by the ratio of the basic element of cell division and auxin
Influence, also influenced by the absolute concentration of two kinds of hormones.4.0 mg·L-1KT and 0.8 mgL-1 The group of the growth substance of NAA
Close the proliferation for being more advantageous to bud.
Four, culture of rootage
After Multiplying culture, by the sprout tuber higher than 2 cm from culture medium B6（With a small amount of callus）It is transferred to 1/2 MS
Rooting induction is carried out in culture medium（Table 5）, 4 pieces of callus of every bottle of inoculation, 1~3 bud of every piece of band, each culture medium inoculated 20
Bottle.Condition of culture：Temperature（25±2）DEG C, relative humidity 40%~60%, intensity of illumination is（33.6±0.7）μmol·m-2·s-1, the photoperiod is that illumination and interlunation are fifty-fifty.
The bud being grown in B6 culture mediums higher than 2 cm is taken to carry out culture of rootage.Find that bud lower part starts point after cultivating 4 weeks
Change forms root.It cultivates the situation of taking root after 8 weeks and is shown in Table 5.Three kinds of rooting induction culture mediums（C1, C2, C3 culture medium are cultivated by basis
The plant growth substance of base and table 5 forms, and the content of wherein basal medium is identical）In, rooting efficiency preferably C1 and C2,
Their rooting rate is 66.75% and 46.85% respectively, and root is more and long；And the rooting rate of C3 culture mediums is relatively low, is 25.30%, root
Also shorter.Table 5 the result shows that, with the raising of KT concentration, rooting rate declines.The best culture medium of rooting induction is C1.
Five, hardening and transplanting
The regrowth obtained in C1 culture mediums is performed physical exercise.Culture bottle is placed at indoor near window and receives scattering
Then natural light irradiation 3 days is opened culture bottle cap, is placed again 3 days in same position.Regrowth is taken out later, is cleaned with clear water
The culture medium of plant base portion is transplanted to containing in 1/3 sand, 2/3 native culture substrate.Other than shade when paying attention in seedling stage, it is not required to
Want special nursing.Radix zanthoxyli growth of seedling after transplanting is good, transplanting survival rate 91%.
Comparative example 1
Experimental method is with embodiment 1, and uniquely the difference is that, culture medium does not contain KT in this comparative example, and callus lures
Conductance only has 1.34~5.31%.
Claims (3)
1. a kind of method of Radix zanthoxyli callus induction, which is characterized in that be using stem section of the Radix zanthoxyli without offspring as explant,
Carry out the induction of callus；The callus inducing medium is MS+1.0~4.0mg/L KT+1.0~2.0mg/L
NAA；
The induction of callus condition is：23~27 DEG C of temperature, relative humidity 40%~60%, intensity of illumination 32.5
~34.5 μm of olm-2·s-1, the photoperiod is that illumination and interlunation are fifty-fifty.
2. the method for Radix zanthoxyli callus induction according to claim 1, which is characterized in that the callus induction training
It is MS+4.0mg/L KT+2.0mg/L NAA to support base.
3. the method for Radix zanthoxyli callus induction according to claim 1, which is characterized in that stem section of the Radix zanthoxyli without offspring
The tender stem segments for taking the Radix zanthoxyli with axillary bud, after disinfection, be inoculated in MS+1.0mg/L~2.0mg/L KT+0.2mg/L~
0.4mg/L NAA culture grow up to 3~4cm without offspring, then intercept the stem section of no offspring.
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|---|---|---|---|---|
| CN102884981B (en) * | 2012-09-26 | 2013-07-24 | 钦州市林业科学研究所 | Zanthoxylum nitidum tissue culture medium |
| CN103651148B (en) * | 2013-12-31 | 2015-09-23 | 茂名市天一农业科技发展有限公司 | The method for tissue culture of a kind of Radix zanthoxyli |
| CN106106190A (en) * | 2016-08-28 | 2016-11-16 | 李志勇 | A kind of tissue cultivation rapid breeding method of Radix Zanthoxyli |
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