CN106508683B - The tissue culture culture medium and cultural method of a kind of Radix zanthoxyli regrowth - Google Patents
The tissue culture culture medium and cultural method of a kind of Radix zanthoxyli regrowth Download PDFInfo
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- OTCCIMWXFLJLIA-BYPYZUCNSA-N N-acetyl-L-aspartic acid Chemical compound CC(=O)N[C@H](C(O)=O)CC(O)=O OTCCIMWXFLJLIA-BYPYZUCNSA-N 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
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Classifications
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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
-
- 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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- Developmental Biology & Embryology (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
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- Environmental Sciences (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a kind of tissue culture culture medium of Radix zanthoxyli regrowth and cultural method；Using stem section of the Radix zanthoxyli without offspring as explant, evoked callus is formed on the MS culture mediums containing suitable KT and other hormone combinations；Using first induction differentiation, then improve differentiation rate and improve the strategy of the quality of bud, the differentiation of induced bud, Radix zanthoxyli regrowth is obtained after rooting induction.The present invention establishes the tissue culturing system that regrowth is obtained by callus phase, improves breeding coefficient（It is existing to realize that the growth coefficient of the method for propagation is 2.4~3.6 by the induction of Multiple Buds, and the growth coefficient of the inventive method can reach more than 5.0）, present invention employs first improving inductivity, then improve the scheme of the quality of bud, so as to obtain high breeding coefficient and the measured seedling of matter, laid 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 tissue culture culture medium of Radix zanthoxyli regrowth
And cultural method.
Background technology
Radix zanthoxyli（Zanthoxylum nitidum (Roxb.) DC.）For Rutaceae xanthoxylum, have very high
Medical value, its 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；
Radix zanthoxyli or the raw material of 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.Because Radix zanthoxyli dosage is huge, its wild resource is almost exhausted, in the market two sides
The adulterant of medicine administered by injection material is therefore also more.Recover the resource of Radix zanthoxyli firstly the need of seedling.It can be sprouted by seed, cutting propagation
Seedling is obtained with tissue cultures.The research of tissue cultures concentrates on the quick breeding of Radix zanthoxyli, that is, use the stem section with axillary bud for
Explant, propagation is realized by the induction of Multiple Buds, but the breeding coefficient of these methods is relatively low.
The content of the invention
The technical problems to be solved by the invention are to overcome drawbacks described above existing for prior art, there is provided a kind of Radix zanthoxyli is again
The tissue culture culture medium of raw seedling.
Second object of the present invention is to provide a kind of tissue culture method of Radix zanthoxyli regrowth.
The purpose of the present invention is achieved by the following technical programs：
Application of the KT in Radix zanthoxyli tissue cultures, the KT is in the medium
Concentration is 0.2~6 mg/L.
Inventor, which studies, to be found to add KT in Radix zanthoxyli（Also known as kinetin, abbreviation KT）It can induce out
Radix zanthoxyli callus, tissue cultures are carried out using callus, growth coefficient can be improved, realize quick breeding.
Inventor, which studies, to be found, Radix zanthoxyli tissue cultures are complex, and explant is different after induction grows up to callus
The species and content of growth substance needed for growth phase are dramatically different, therefore the invention provides a set of from callus induction
To the culture medium taken root, that is, provide a kind of tissue culture culture medium of Radix zanthoxyli regrowth, including callus inducing medium, bud point
Change culture medium, bud proliferated culture medium and root media；The callus inducing medium is the mg/L KT of MS+1.0~4.0
+ 1.0~2.0 mg/LNAA；The bud differential medium is divided into first time bud differential medium and second of bud differentiation culture
Base, the first time bud differential medium are the mg/L of mg/L KT+0.1 of MS+1.0~4.0~0.2 IBA, described second
Bud differential medium is the mg/L KT+0.4 of MS+2.0~6.0 mg/L KT+0.2 mg/L IBA or MS+2.0~6.0
~0.8 mg/L NAA；The bud proliferated culture medium is MS+2.0~4.0 mg/L KT+0.8 mg/L IBA or MS+
The mg/L NAA of 2.0~4.0 mg/L KT+0.4~0.8；The root media is the mg/L KT+ of 1/2MS+0.2~2.0
1.0 mg/L IBA。
Preferably, the callus inducing medium is MS+4.0 mg/L KT+2.0 mg/LNAA；The bud differentiation
Culture medium is MS+6.0 mg/L KT+0.2 mg/L IBA；The bud proliferated culture medium is MS+4.0 mg/L KT+0.8
mg/L NAA；The root media is 1/2 MS+0.2 mg/L KT+1.0 mg/L IBA.
The present invention also provides a kind of method of Radix zanthoxyli tissue cultures, comprises the following steps：
（1）Using stem section of the Radix zanthoxyli without offspring as explant, the induction of callus is carried out；The callus induction training
It is the mg/LNAA of mg/L KT+1.0 of MS+1.0~4.0~2.0 to support base；
（2）Callus is subjected to differentiation culture at twice in differential medium, to obtain band bud callus；It is described
First time bud differential medium is the mg/L IBA of mg/L KT+0.1 of MS+1.0~4.0~0.2, second of bud differentiation
Culture medium is mg/L KT+0.4~0.8 of MS+2.0~6.0 mg/L KT+0.2 mg/L IBA or MS+2.0~6.0
mg/L NAA；
（3）Callus with bud is cultivated in bud proliferated culture medium, to obtain more regeneration buds, and improves bud
Quality；The bud proliferated culture medium is the mg/ of MS+2.0~4.0 mg/L KT+0.8 mg/L IBA or MS+2.0~4.0
The mg/L NAA of L KT+0.4~0.8；
（4）Gained bud is cut from base portion, is transferred to root induction in root media；The root media is 1/2
The mg/L KT+1.0 mg/L IBA of MS+0.2~2.0.
Preferably, step（1）、（2）、（3）、（4）Condition of culture be：23~27 DEG C of temperature, relative humidity 40%~
60%, intensity of illumination is 32.5~34.5 μm of olm-2·s-1, the photoperiod is illumination and interlunation half and half.
Preferably, step（1）The preparation method of stem section of the Radix zanthoxyli without offspring be：Take the young tender of the Radix zanthoxyli with axillary bud
Stem section, after sterilization, it is inoculated in the nothing that the mg/L NAA cultures of mg/L KT+0.2 of MS+1.0~2.0~0.4 grow up to 3~4cm
Offspring, then intercept the stem section of no offspring.
Compared with prior art, the invention has the advantages that：
The present invention induces using stem section of the Radix zanthoxyli without offspring as explant on the MS culture mediums matched containing optimum hormone
Callus formation；Using first induction differentiation, then improve differentiation rate and improve the strategy of the quality of bud, the differentiation of induced bud, warp
Radix zanthoxyli regrowth is obtained after rooting induction.The present invention establishes the tissue cultures body that regrowth is obtained by callus phase
System, present invention employs first improving inductivity, then improve the scheme of the quality of bud, so as to obtain high breeding coefficient and quality
Good seedling, existing to realize that the growth coefficient of the method for propagation is 2.4~3.6 by the induction of Multiple Buds, side of the invention
The breeding coefficient of method Radix zanthoxyli reaches 5.0, higher than the breeding coefficient of rapid propagation method, if squamous subculture callus 1 time,
Then breeding coefficient is doubled to 10.0, if squamous subculture callus 2 times, breeding coefficient 20.0, this is quick breeding side
Method without advantage, culture medium and cultural method of the present invention lay the foundation for a large amount of plantation Radix zanthoxylis.
Embodiment
Present disclosure is further illustrated 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, the simple modifications or substitutions made to the inventive method, step or condition,
Belong 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 meanses.
Vegetable material：Radix zanthoxyli is without offspring stem section.Radix zanthoxyli tender stem segmentses with axillary bud pick up from Traditional Chinese Medicine University Of Guangzhou's medicine
With botanical garden, soaked 30 seconds with 70% ethanol, then use 0.1%HgCl2Immersion 8 minutes, with 0.5%NaClO soak 10 minutes, finally
With aseptic water washing 3 times, MS+2.0 mgL are inoculated in-1KT+0.4 mgL-1Cultivated in NAA, start to give birth to after 12 days
It is long, about 12 Zhou Houke grow up to 3~4 cm without offspring.
The culture medium based on MS culture mediums, addition sucrose 30 gL-1, the gL of carragheen 8.0-1, pH5.8~6.0 are made
Based on culture medium.Plant growth substance includes α-naphthylacetic acid（α-naphthalene acetic acid, NAA）, indoles fourth
Acid（Indole-3-butyric acid, IBA）, KT（Kinetin, KT）.
Callus induction rate：Respectively in stem section callus Fiber differentiation the 10th, 20,30 day, callus growth feelings are observed
Condition, count 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 propagation bud number/inoculation bud number).
Rooting rate：Culture of rootage 8 weeks, count rooting rate（Rooting rate=(the bud number for the bud number/inoculation taken root) × 100%）.
Embodiment 1
First, the induction of callus
Stem without offspring is aseptically cut into about 6 mm segment, 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 every kind of 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 illumination and interlunation half and half, a month subculture 1 time.
The inducing culture that Radix zanthoxyli stem section is formed 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 produce callus group
Knit, generation callus can be induced by illustrating the KT and NAA of debita spissitudo.Compare the effect that three kinds of culture mediums produce callus
Fruit, the inductivity highest in A3 culture mediums, reaches 74.84%；Next to that A1 culture mediums, inductivity 66.84%；Worst is
A2 culture mediums, inductivity only have 58.76%.The quality of caused callus also has difference, the caused callus group on A3 and A1
The color knitted is faint yellow, graininess, quality is loose and moistens；And the color of caused callus is yellowish on A2
Color, bulk, quality are stiff；Therefore caused callus ratio is convenient as callus differentiation on A3 and A1 culture mediums
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.
2nd, 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
Support in base（Table 2, culture medium B1, B2, B3 be made up 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 every kind of culture medium., will be caused by culture medium B3 more after culture 1 month
Injured tissue is then transferred in another 4 kinds of differential mediums and cultivated（Table 3）, each 8 pieces of culture dish inoculation, the respectively inoculation 12 of every kind of culture medium
Individual 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 illumination and interlunation half and half.
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
Have differentiation sprout the reason for be probably KT concentration it is too low.From the results shown in Table 2, in three kinds of culture mediums, containing 4 mg/L
KT+0.2 mg/L IBA B3 culture mediums have certain effect to callus differentiation, but effect is preferable not enough.In order to enter
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
Into 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, B4 KT
Content highest, the bud that differentiates is smaller, color is partially yellow, blade is tiny, illustrates the second-rate of bud.Therefore the result of table 3 shows,
Differentiation of the KT of high concentration to bud is favourable, but KT concentration is too high, can make the degradation of bud.
The interpretation of result of consolidated statement 2 and table 3, bud differentiation culture in, except KT concentration can not it is too low in addition to, auxin
Concentration also has an impact to the differentiation rate of bud, and in the processing of KT concentration identical, auxin concentration is high, then has inductivity is high to become
Gesture.In addition, the inductivity of the B3 culture mediums of table 3 is higher than the inductivity of the B3 culture mediums in table 2, reason is probably table 3
The incubation time of callus is longer.
3rd, the propagation 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, every kind of culture medium is respectively inoculated with 8 culture dishes.4 weeks follow-up generations 1
It is secondary, it is inoculated into blake bottle, the callus of every bottle of inoculation 3 pieces of bands, 1~2 bud, respectively 16 bottles of the inoculation of every kind of culture medium.Cultivate bar
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 and interlunation half and half.
The differentiation rate of bud is apparently higher than other culture mediums in B4 culture mediums, but bud is second-rate, it is possible the reason for be 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
Newly it is 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, its bud growth coefficient were 3.33 respectively to bud cultivation effect with after 8 weeks（Culture 4 weeks）With 5.00（Training
Support 8 weeks）.Next to that 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
Green, the quality for illustrating bud is good.Cultivation effect on B5 medium is worst, and its KT concentration is 2.0 mgL-1.So
In Multiplying culture, the too low propagation for being unfavorable for bud of KT concentration.
Comparing the plant growth substance composition in B5 and B6 culture mediums, their KT and NAA concentration proportion are all 5, but
The cultivation effect of both buds is different, and 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 basic element of cell division and the ratio of auxin
Influence, also influenceed by the absolute concentration of two kinds of hormones.4.0 mg·L-1KT and 0.8 mgL-1 The group of NAA growth substance
Close the propagation for being more beneficial for bud.
4th, 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, every kind of 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 illumination and interlunation half and half.
The bud being grown in B6 culture mediums higher than 2 cm is taken to carry out culture of rootage.Find that bud bottom starts point after cultivating 4 weeks
Change forms root.Cultivate the situation of taking root after 8 weeks and be 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；It is 25.30% and the rooting rate of C3 culture mediums is relatively low, root
Also it is shorter.The result of table 5 shows that, with the rise of KT concentration, rooting rate declines.The best culture medium of rooting induction is C1.
5th, hardening and transplanting
The regrowth obtained in C1 culture mediums is performed physical exercise.Blake bottle is placed at indoor near window and receives scattering
Natural light irradiation 3 days, culture bottle cap is then opened, is placed again 3 days in same position.Regrowth is taken out afterwards, is cleaned with clear water
The culture medium of plant base portion, it is transplanted to containing in 1/3 sand, 2/3 native culture matrix.When paying attention in seedling stage in addition to shade, 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 with embodiment 1, it is unique unlike：In the step of embodiment 1 one in callus inducing medium
KT is not contained, its inductivity is 1.34%~5.31%.
Claims (4)
1. a kind of tissue culture culture medium of Radix zanthoxyli regrowth, it is characterised in that including callus inducing medium, bud differentiation training
Support base, bud proliferated culture medium and root media；The callus inducing medium is MS+1.0~4.0mg/L KT+1.0
~2.0mg/L NAA；The bud differential medium is divided into first time bud differential medium and second of bud differential medium, and first
Secondary bud differential medium is MS+1.0~4.0mg/L KT+0.1~0.2mg/L IBA, and second of bud differential medium is MS+
2.0~6.0mg/L KT+0.2mg/L IBA or MS+2.0~6.0mg/L KT+0.4~0.8mg/L NAA；The bud propagation
Culture medium is MS+2.0~4.0mg/L KT+0.8mg/L IBA or MS+2.0~4.0mg/L KT+0.4~0.8mg/L
NAA；The root media is 1/2MS+0.2~2.0mg/L KT+1.0mg/L IBA.
A kind of 2. method of Radix zanthoxyli tissue cultures, it is characterised in that comprise the following steps：
(1) using stem section of the Radix zanthoxyli without offspring as explant, the induction of callus is carried out；The callus inducing medium
For MS+1.0~4.0mg/L KT+1.0~2.0mg/L NAA；
(2) callus is subjected to differentiation culture at twice in differential medium, to obtain band bud callus；Described first
Secondary bud differential medium is MS+1.0~4.0mg/L KT+0.1~0.2mg/L IBA, and second of bud differential medium is
MS+2.0~6.0mg/L KT+0.2mg/L IBA or MS+2.0~6.0mg/L KT+0.4~0.8mg/L NAA；
(3) callus with bud is cultivated in bud proliferated culture medium, to obtain more regeneration buds, and improves the matter of bud
Amount；The bud proliferated culture medium is MS+2.0~4.0mg/L KT+0.8mg/L IBA or MS+2.0~4.0mg/L KT+0.4
~0.8mg/L NAA；
(4) gained bud is cut from base portion, is transferred to root induction in root media；The root media is 1/2MS+
0.2~2.0mg/L KT+1.0mg/L IBA.
3. the method for Radix zanthoxyli tissue cultures according to claim 2, it is characterised in that step (1), (2), (3), (4)
Condition of culture is：23~27 DEG C of temperature, relative humidity 40%~60%, intensity of illumination are 32.5~34.5 μm of olm-2·s-1, the photoperiod is illumination and interlunation half and half.
4. the method for Radix zanthoxyli tissue cultures according to claim 2, it is characterised in that the Radix zanthoxyli of step (1) is without offspring
Stem section be to take the tender stem segmentses of the Radix zanthoxyli with axillary bud, after sterilization, be inoculated in MS+1.0~2.0mg/L KT+0.2~
0.4mg/L NAA cultures grow up to 3~4cm without offspring, then intercept the stem section of no offspring.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102884981A (en) * | 2012-09-26 | 2013-01-23 | 钦州市林业科学研究所 | Zanthoxylum nitidum tissue culture medium |
| CN103651148A (en) * | 2013-12-31 | 2014-03-26 | 茂名市天一农业科技发展有限公司 | Tissue culturing method 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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|---|---|---|---|---|
| CN102884981A (en) * | 2012-09-26 | 2013-01-23 | 钦州市林业科学研究所 | Zanthoxylum nitidum tissue culture medium |
| CN103651148A (en) * | 2013-12-31 | 2014-03-26 | 茂名市天一农业科技发展有限公司 | Tissue culturing method 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 |
Non-Patent Citations (2)
| Title |
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| 两面针的组织培养与快速繁殖;王小敏等;《玉林师范学院学报（自然科学）》;20051231;第26卷(第5期);第70-73页 * |
| 两面针的组织培养研究;莫勇生等;《农业研究与应用》;20121231(第6期);第16-20页 * |
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