CN105432472B - A kind of method of tuniclike psammosilene root adventitious root fast breeding culture - Google Patents
A kind of method of tuniclike psammosilene root adventitious root fast breeding culture Download PDFInfo
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Abstract
A kind of method of tuniclike psammosilene root adventitious root fast breeding culture, belong to the method for plant organ hyperblastosis culture.Tuniclike psammosilene root adventitious root tissue block is inoculated in the solid medium of MS+1.0mg/L IBA+30g/L white granulated sugars or MS+1.0mg/L KT+30g/L white granulated sugars by the present invention cultivates adventitious root, caused indefinite root dry weight and dry rate are higher wherein in the culture medium of MS+1.0mg/L KT+30g/L white granulated sugars, and during using carbon source of the 30g/L white granulated sugars as MS solid mediums, its dry rate is better than glucose, and cost is less than sucrose.Conditions above is in can obtain more rich tuniclike psammosilene root adventitious root in 40d.The present invention can put into practice for industrialization production and provide foundation and guidance.
Description
Technical field
The invention belongs to plant organ tissue cultures, particularly medicinal plant adventitious root culture medium.
Background technology
Tuniclike psammosilene root（Psammosilene tunicoides W.C.Wu et C.Y.Wu）For Caryophyllaceae
（Caryophyllaceae）Tuniclike psammosilene root category（Psammosilene）The perennial medicinal herb plant of autogenus, also known as Kunming the root of straight ladybell,
RADIX PSAMMOSILENE etc..Tuniclike psammosilene root root contains saponin constituent, and is used as medicine containing amino acid, organic acid, triterpene etc., its root after drying, and is that Yunnan is white
The main component of medicine, short-pedicel aconite root, pain Urapidil, golden bone lotus capsule etc..China lacks natural resources of Chinese medicinal materials effective protection measure,
Wild resource is endangered, and the scarcity of plant resources can be made up with artificial culture and industrialization production.The training of tuniclike psammosilene root adventitious root
Support mainly is influenceed by several respects such as explant, inoculum concentration, plant hormone, sucrose and inorganic salts.In relevant report, Zhao Shuanhe
Li Jingbin was once utilized respectively agrobacterium rhizogenes tuniclike psammosilene root and obtains hairy root, and studied the optimal culture conditions of tuniclike psammosilene root hairy root.
But the technology is needed compared with high-tech and appointed condition, still there is limitation.（The Primary Study of the tuniclike psammosilene root hairy root inductions such as Zhao Shuan
[J] Chinese medicines, 2012,35（2）：176-179；Foundation [J] of the such as Li Jingbin tuniclike psammosilene root hairy root inductions and cultivating system
CHINA JOURNAL OF CHINESE MATERIA MEDICA, 2011,36 (5): 547-551）.Document：" influence tuniclike psammosilene root growth of hair root and saponin(e accumulation because
Element " is to induce hairy root with agrobacterium rhizogenes ACCC10060 strain infection tuniclike psammosilene roots stem section, blade, is sieved through anti-kanamycins
Choosing culture, the hairy root of acquisition is adventitious root Liquid Culture material, is inoculated with 5 kinds of fluid nutrient mediums that with the addition of exogenous hormone respectively
In, tuniclike psammosilene root adventitious root is bred preferably with adding NAA, IAA, IBA 1/2MS fluid nutrient mediums, and find growth rate and soap
Glycosides yield is proportionate.（Tian Sidi etc., when treasure's traditional Chinese medical science traditional Chinese medicines [J], 2012,23 (4):824-826.）.Another document：It is " different
Influence of the condition of culture to tuniclike psammosilene root growth of hair root "（Gao Shuai, Wang Hongfeng, Hou Lili, etc., Annual [J],
2012, 28(2):16-21.）It is optimal medium to select 1/2MS solid mediums, the adventitious root after being infected with agrobacterium rhizogenes
Cultivated for material.
Sugar is that explant grows to form the element of carbon skeleton, while maintains the osmotic pressure of culture medium.Different types of sugar,
Molecular structure is different, and its form is more obvious to adventitious root growth effect with concentration.Two documents of the above think sucrose as training
The carbon source of base is supported, the accumulation of tuniclike psammosilene root hairy root biomass is maximum.
The content of the invention
It is contemplated that establish a kind of method of tuniclike psammosilene root adventitious root fast breeding culture, it include by minimal medium and
Its culture medium system formed with variety classes hormone combinations, in order to select corresponding culture medium to be given birth to according to produce reality
Production, so as to reduce production cost, efficiency is improved, guidance is provided for factorial praluction tuniclike psammosilene root adventitious root.
The present invention realizes in following methods：
（1）The terminal bud for cutting tuniclike psammosilene root aseptic seedling is inoculated in 1/2MS+0.3mg/L IBA+0.1mg/L NAA+ 0.3g/L
In the root media of activated carbon, the indefinite network of roots of hairy is formed after 60d；
（2）By block mode cut expand it is numerous after the indefinite network of roots of tuniclike psammosilene root, be inoculated in the MS that with the addition of hormone and white granulated sugar
In solid minimal medium, every bottle of packing 25mL culture medium, 4 pieces of indefinite networks of roots of tuniclike psammosilene root of every bottle of inoculation.
Further, described culture medium is one below kind：
（1）The combination of MS solids minimal medium and auxin：The mg/L IBA of MS+30 g/L white granulated sugars+1.0；
（2）The combination of MS solids minimal medium and the basic element of cell division：The mg/L KT of MS+30 g/L white granulated sugars+1.0；
More than（1）~（2）Condition of culture is:25 ± 2 DEG C, the h/d of light application time 12, the lx of intensity of illumination 1500~2000,
Incubation time 40d.
Preferably, described culture medium is the combination of MS solids minimal medium and the basic element of cell division：MS+1.0 mg/L
KT。
Further, described culture medium is：The tuniclike psammosilene root adventitious root tissue block for being inoculated in described culture medium is 0.05g/
Block, the tissue block dry weight are roughly equal to 0.0056 ± 0.0004g/ blocks.
The marked improvement of the present invention is：
（1）The present invention is thought by the medium culture result of 2 kinds of different constituents：Except the gene for considering selected materials
Type is different outer, minimal medium that should be using MS solid mediums as tuniclike psammosilene root hairy root, under the minimal medium, even if not
Its fresh weight of addition hormone can also reach high value, and this is different from being typically chosen 1/2MS culture mediums at present.
（2）Take sucrose and glucose different from prior art, the present invention considers from indefinite root growth effect and production cost：
The white granulated sugar that 30 g/L are added in MS culture mediums is more suitable for growth and the biomass accumulation of tuniclike psammosilene root adventitious root.In 30 g/L
White granulated sugar under, do not add the high value that hormone its fresh weight can reach 5.088 g, its dry weight is 0.480g, and higher number
Value.It reflects：Supplementary carbon source of the white granulated sugar as culture medium, with 30 g/L dosage, cost can be effectively reduced, and can obtains
Obtain higher hairy root output.
（3）The present invention adds 1.0 mg/L IBA in the MS minimal mediums for the addition of 30g/L white granulated sugars and cultivates golden iron
Adventitious root is locked, can also obtain preferable effect.
（4）The index of the evaluation indefinite root growth situation of tuniclike psammosilene root includes fresh weight, the increment of dry weight and dry rate.Wherein,
Dry rate refers to the dry weight of adventitious root and the ratio of fresh weight, and it reflects the net yield of adventitious root.It with the addition of 30g/L white granulated sugars
MS solid mediums on, the present invention changes the plant hormones of tuniclike psammosilene root tissue cultures, is adding 1.0 mg/L KT MS trainings
Indefinite root dry weight and dry rate highest in base is supported, and it is notable with other processing differences, KT is more beneficial for the life of tuniclike psammosilene root adventitious root
It is long.Relative to the data of existing literature, tuniclike psammosilene root adventitious root that the solid medium of the MS+1.0 mg/L KT is obtained it is dry
Thing rate is 11.96%, and its fresh weight is 173.2 gL-1, dry weight is 20.72 gL-1, dry weight is 1.52 times of existing literature.
Brief description of the drawings
Fig. 1 is indefinite root growth situation in WPM minimal mediums.
Fig. 2 is indefinite root growth situation in MS minimal mediums.
Fig. 3 is indefinite root growth situation in the culture medium of MS+30 g/L white granulated sugars.
Fig. 4 is indefinite root growth situation in the mg/L culture mediums of MS+IBA 1.0.
Fig. 5 is indefinite root growth situation in the mg/L culture mediums of MS+KT 1.0.
Fig. 6 is indefinite root growth situation in the mg/L culture mediums of 0.5 mg/L+KT of MS+ NAA 0.5.
Above MS refers to MS solid mediums, and KT refers to kinetin, and IBA refers to 3- indolebutyric acids, and NAA refers to α-naphthalene second
Acid.
The present invention will be further described with reference to embodiments.The present invention includes but is not limited to the content of embodiment.
Embodiment
1 materials and methods
1.1 material
Aseptically, the terminal bud for cutting tuniclike psammosilene root aseptic seedling is inoculated in 1/2MS+0.3mg/L IBA+0.1mg/L
Culture of rootage in the root media of NAA+ 0.3g/L activated carbons, after 60 days, the indefinite network of roots of hairy is trained, it is standby.
1.2 method
During inoculation, cut above-mentioned indefinite network of roots by block mode and cultivated.
1.2.1 influence of the different minimal mediums to the indefinite root biomass of tuniclike psammosilene root
Tuniclike psammosilene root adventitious root tissue block, every piece of about 0.05g are cut, dry weight is roughly equal to 0.0056 ± 0.0004g/ blocks, connect respectively
Kind often handles 5 bottles, every bottle of packing in the addition of respectively in tri- kinds of solid minimal mediums of MS, 1/2MS, WPM of 30 g/L sucrose
25mL culture mediums, every bottle is inoculated with 4 pieces, is repeated 3 times, and fresh weight, the dry weight of tuniclike psammosilene root adventitious root are determined when 40d.Condition of culture
For:25 ± 2 DEG C of temperature, the h/d of light application time 12, the lx of intensity of illumination 1500~2000.Similarly hereinafter.
1.2.2 influence of the various concentrations carbon source kind to the indefinite root biomass of tuniclike psammosilene root
Tuniclike psammosilene root adventitious root tissue block, every piece of about 0.05g are cut, dry weight is roughly equal to 0.0056 ± 0.0004g/ blocks, connect respectively
Kind is on the minimal medium of different carbon source.The culture medium be separately added into concentration be 20,30 and 40 g/L white granulated sugar, sucrose and
Glucose, experiment amount to 9 kinds of processing, often handle 5 bottles, every bottle of packing 25mL culture medium, every bottle connects 4 pieces, is repeated 3 times, in the 40th
Fresh weight, the dry weight of tuniclike psammosilene root adventitious root are determined during d.
1.2.3 influence of the various concentrations auxin to the indefinite root biomass of tuniclike psammosilene root
Minimal medium and the carbon source for screening to obtain based on 1.2.1 and 1.2.2, add IBA the and NAA conducts of various concentrations
It addition of the culture medium of auxin.
Tuniclike psammosilene root adventitious root tissue block, every piece of about 0.05g are cut, its dry weight is roughly equal to 0.0056 ± 0.0004g/ blocks, respectively
It is inoculated on the culture medium that addition of auxin.Experiment amounts to 9 kinds of medium treatments.5 bottles are often handled, every bottle of packing 25mL culture
Base, every bottle connects 4 pieces, repeat number 3, and the fresh weight and dry weight of tuniclike psammosilene root adventitious root are determined when 40 d.
1.2.4 influence of the various concentrations basic element of cell division to the indefinite root biomass of tuniclike psammosilene root
Minimal medium and the carbon source for screening to obtain based on 1.2.1 and 1.2.2, add KT, 6-BA and TDZ of various concentrations
As the culture medium that addition of the basic element of cell division.
Tuniclike psammosilene root adventitious root tissue block, every piece of about 0.05g are cut, its dry weight is roughly equal to 0.0056 ± 0.0004g/ blocks, respectively
It is inoculated on the culture medium that addition of the basic element of cell division.Experiment amounts to 11 kinds of medium treatments, often handles 5 bottles, every bottle of packing
25mL culture mediums, every bottle connects 4 pieces, repeat number 3, and the fresh weight and dry weight of tuniclike psammosilene root adventitious root are determined when 40 d.
1.2.5 hormon is with the influence for comparing the indefinite root biomass of tuniclike psammosilene root
Minimal medium and the carbon source for screening to obtain based on 1.2.1 and 1.2.2, add KT, 6-BA of various concentrations, IBA,
NAA and TDZ is as the culture medium that addition of exogenous hormone.
Tuniclike psammosilene root adventitious root tissue block, every piece of about 0.05g are cut, its dry weight is roughly equal to 0.0056 ± 0.0004g/ blocks, respectively
It is inoculated on the culture medium that addition of exogenous hormone, experiment designs 8 kinds of processing culture mediums altogether, often handles 5 bottles, every bottle of packing 25mL
Culture medium, every bottle is inoculated with 4 pieces, is repeated 3 times, and the fresh weight and dry weight of tuniclike psammosilene root adventitious root are determined when 40 d.
1.3 data analysis
The data obtained carries out statistical analysis in SPSS17.0 and the softwares of EXCELL 2007, wherein：
Fresh weight：The weight of fresh adventitious root in 1 bottle of culture medium
Dry weight：Weight in 1 bottle of culture medium after fresh adventitious root drying
Dry rate=dry weight/fresh weight
Sugared dosage in every gram of sugared actuating quantity=1 liter culture in the dry weight incrementss/1 liter culture medium of adventitious root
2 results and analysis
Influence of the 2.1 different minimal mediums to the indefinite root biomass of tuniclike psammosilene root
Tuniclike psammosilene root adventitious root is inoculated in MS, 1/2MS, WPM culture medium respectively, and inoculation 4d can grow a little tender white
Color adventitious root.It the results are shown in Table 1.
Influence of the 1 different minimal mediums of table to the indefinite root biomass of tuniclike psammosilene root
| Kinds of culture medium | Fresh weight（g） | Dry weight（g） | Dry rate % | Root growth situation |
| MS | 4.968a | 0.474a | 9.54a | Branch is more and thick, faint yellow |
| 1/2MS | 4.018a | 0.361b | 8.98b | Branch is more and thick, faint yellow |
| WPM | 3.599b | 0.359c | 9.97a | Branch is more and thin, aubergine |
Note：The different letters of same column represent the significant difference in 0.05 level in table, similarly hereinafter.
As shown in Table 1, in 3 kinds of culture mediums, adventitious root branch is more, fresh with its from the indefinite root growth situation of tuniclike psammosilene root
Weight, the growth of dry weight and dry rate are evaluated, it is believed that MS is the more excellent minimal medium of the indefinite root growth of tuniclike psammosilene root（Fig. 1）.
Influence of the 2.2 various concentrations carbon source kinds to the indefinite root biomass of tuniclike psammosilene root
In MS minimal mediums, various concentrations and different types of carbon source are added, tuniclike psammosilene root adventitious root biology after inoculation
The accumulation of amount is shown in Table 2.
The influence of the different carbon source species of table 2 and concentration to the indefinite root biomass of tuniclike psammosilene root
As seen from Table 2, when cultivating 40d, when concentration is 30g/L and 40g/L, its fresh weight and dry weight are equal for white granulated sugar and sucrose
It is higher, and difference is not notable（Respectively 5.088 g and 0.48 g, 4.899 g and 0.392 g, 5.158 g and 0.486 g with
And 5.956 g and 0.515 g）；And the culture medium using glucose as carbon source, the accumulation effect of tuniclike psammosilene root adventitious root are poor.From every
From the point of view of gram sugared action effect, 20 g/L, 30 g/L sucrose and 30 g/L white granulated sugars pair respectively are added in MS minimal mediums
Without significant difference between the action effect of adventitious root biomass accumulation, action effect it is worst be the MS for adding 40 g/L glucose
Culture medium, its sugared actuating quantity is only 0.250.Because in the case of the same dosage of carbon source, the cost of sucrose will be far above in vain
Granulated sugar, therefore, binding tests result, and consider that 30 g/L white granulated sugars are tuniclike psammosilene root adventitious root from the economic angle of production cost
The most suitable carbon source cultivated on MS culture mediums（Fig. 2）.
Influence of the 2.3 various concentrations auxin to the indefinite root biomass of tuniclike psammosilene root
In it addition of the MS culture mediums of various concentrations auxin respectively, tuniclike psammosilene root adventitious root biomass accumulation situation is shown in Table
3。
Influence of the various concentrations auxin of table 3 to the indefinite root biomass of tuniclike psammosilene root
Table 3 shows that after the d of tuniclike psammosilene root adventitious root culture 40, when IBA concentration is 1.0 mg/L, indefinite root growth is preferable, this
The fresh weight of the lower adventitious root of processing is 4.754 g, and dry weight is 0.504 g, is all remarkably higher than other processing, and respectively about compare
（2.918 g and 0.337 g）1.5 times；But when IBA concentration is 3.0 mg/L, tuniclike psammosilene root adventitious root biomass dry weight is only
0.306 g, for the minimum in all processing.It can be seen that suitable exogenous auxin and its concentration are the indefinite root growths of tuniclike psammosilene root
Requirement.For table 3 it is also shown that when culture medium is 1.0 mg/L of MS+IBA, its dry rate also shows as 10.60% compared with Gao Shui
It is flat.The indefinite root biomass growth pattern of tuniclike psammosilene root is considered, is being attached with the MS of 1.0 mg/L IBA+30g/L white granulated sugars
In solid medium, the indefinite root growth of tuniclike psammosilene root is preferable（Fig. 3）.
Influence of the 2.4 various concentrations basic elements of cell division to the indefinite root biomass of tuniclike psammosilene root
Tuniclike psammosilene root adventitious root is inoculated in the MS solid mediums for being attached with the various concentrations basic element of cell division, observes and surveys
Its fixed indefinite root biomass increase situation.
Influence of the various concentrations basic element of cell division of table 4 to the indefinite root growth of tuniclike psammosilene root
After adventitious root culture 40d, 1.0 mg/L KT, 0.01 mg/L 6-BA and 0.001 mg/L TDZ MS are attached with
Indefinite root fresh weight is higher in culture medium, and without significant difference between three；In additional 0.3 mg/L 6-BA culture medium,
Indefinite root fresh weight is minimum, only 0.619 g；In the mg/L KT of MS+ 1.0 culture medium, indefinite root dry weight highest, it is
0.518g, and handle equal significant difference with other.And the minimum culture medium of gained dry weight is the mg/L 6-BA of MS+ 0.3, the training
The dry weight for supporting adventitious root in base is only 0.094g.
In additional 1.0 mg/L KT culture mediums, the adventitious root dry rate of higher level is obtained（Table 4）, and adventitious root
Growth is preferably (Fig. 4).Therefore, in the solid medium of MS+1.0 mg/L KT+30g/L white granulated sugars, the life of tuniclike psammosilene root adventitious root
It is long preferable.Calculated with dry weight 0.518g/25mL, the dry weight for the tuniclike psammosilene root adventitious root that MS solid mediums are obtained is 20.72
g·L-1, it is document " factor for influenceing tuniclike psammosilene root growth of hair root and saponin(e accumulation "（Tian Sidi etc., when treasure's traditional Chinese medical science traditional Chinese medicines [J],
2012,23 (4):824-826.）1.52 times of report.
（Note：According to document above, its 1/2MS+0.5mg/L IBA dry weight yield highest, is 13.67g/L.With the present invention
Compare, in MS+1.0 mg/L KT solid medium, its dry weight is 0.518g per 25mL, then 1L dry weight is 0.518 g/
25mL×（40×25mL）=20.72g, therefore the ratio between the present invention and document dry weight yield：20.72g/13.67g=1.52 times.）
2.5 hormons are with the influence for comparing the indefinite root biomass of tuniclike psammosilene root
Tuniclike psammosilene root adventitious root is inoculated in the solid medium matched containing different auxin with the basic element of cell division and cultivated, its
As a result it is listed in table 5.
Influence of the different culture media scheme of table 5 to the indefinite root growth of tuniclike psammosilene root
As can be seen from Table 5, certain difference, wherein dry weight be present most in the 40th d after incubation, the dry weight of 8 combinations
It is low to combine the mg/L NAA+30g/L white granulated sugars of MS+0.01 mg/LTDZ+0.05, its dry weight is only 0.224g；And combine
The mg/L KT+30g/L white granulated sugars of MS+0.5 mg/LNAA+0.5, its dry weight are 0.474g（Fig. 5）, for the maximum of 8 combinations
Value, but the output respectively less than in MS+1.0 mg/L KT+30g/L white granulated sugars and 1.0 mg/L IBA+30g/L white granulated sugars.
Claims (3)
1. a kind of method of tuniclike psammosilene root adventitious root fast breeding culture, including:
(1) terminal bud for cutting tuniclike psammosilene root aseptic seedling is inoculated in 1/2MS+0.3mg/L IBA+0.1mg/L NAA+0.3g/L activity
In the root media of charcoal, the indefinite network of roots of hairy is formed after 60d；
It is characterized in that：
（2）By block mode cut expand it is numerous after the indefinite network of roots of tuniclike psammosilene root, be inoculated in the MS solids that with the addition of hormone and white granulated sugar
In minimal medium, every bottle of packing 25mL culture medium, 4 pieces of indefinite networks of roots of tuniclike psammosilene root of every bottle of inoculation；
Described culture medium is one below kind：
（1）The combination of MS solids minimal medium and auxin：The mg/L IBA of MS+30 g/L white granulated sugars+1.0；
（2）The combination of MS solids minimal medium and the basic element of cell division：MS+30g/L white granulated sugars+1.0mg/LKT；
More than（1）~（2）Condition of culture is：25 ± 2 DEG C, light application time 12h/d, 1500 ~ 2000lx of intensity of illumination, incubation time
40d。
2. the method for tuniclike psammosilene root adventitious root fast breeding culture according to claim 1, it is characterized in that：Described culture medium
For the combination of MS solids minimal medium and the basic element of cell division：MS+1.0 mg/L KT.
3. the method for tuniclike psammosilene root adventitious root fast breeding culture according to claim 1 or 2, it is characterized in that：It is inoculated in institute
The tuniclike psammosilene root adventitious root tissue block for the culture medium stated is 0.05g/ blocks, and the tissue block dry weight is 0.0056 ± 0.0004 g/ blocks.
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