CN106342689B - A kind of South America astral oil rattan rapid propagation method - Google Patents
A kind of South America astral oil rattan rapid propagation method Download PDFInfo
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- CN106342689B CN106342689B CN201610803352.XA CN201610803352A CN106342689B CN 106342689 B CN106342689 B CN 106342689B CN 201610803352 A CN201610803352 A CN 201610803352A CN 106342689 B CN106342689 B CN 106342689B
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- 241000345998 Calamus manan Species 0.000 title claims abstract description 60
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- 230000006698 induction Effects 0.000 claims abstract description 59
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- 239000001963 growth medium Substances 0.000 claims description 29
- 239000012883 rooting culture medium Substances 0.000 claims description 26
- 229930006000 Sucrose Natural products 0.000 claims description 24
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 24
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- 229910000359 iron(II) sulfate Inorganic materials 0.000 description 4
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- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 description 4
- 229910000357 manganese(II) sulfate Inorganic materials 0.000 description 4
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 4
- 239000011684 sodium molybdate Substances 0.000 description 4
- TVXXNOYZHKPKGW-UHFFFAOYSA-N sodium molybdate (anhydrous) Chemical compound [Na+].[Na+].[O-][Mo]([O-])(=O)=O TVXXNOYZHKPKGW-UHFFFAOYSA-N 0.000 description 4
- UEUXEKPTXMALOB-UHFFFAOYSA-J tetrasodium;2-[2-[bis(carboxylatomethyl)amino]ethyl-(carboxylatomethyl)amino]acetate Chemical compound [Na+].[Na+].[Na+].[Na+].[O-]C(=O)CN(CC([O-])=O)CCN(CC([O-])=O)CC([O-])=O UEUXEKPTXMALOB-UHFFFAOYSA-J 0.000 description 4
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 description 4
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- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 2
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- MSSXOMSJDRHRMC-UHFFFAOYSA-N 9H-purine-2,6-diamine Chemical compound NC1=NC(N)=C2NC=NC2=N1 MSSXOMSJDRHRMC-UHFFFAOYSA-N 0.000 description 1
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- TWFZGCMQGLPBSX-UHFFFAOYSA-N Carbendazim Natural products C1=CC=C2NC(NC(=O)OC)=NC2=C1 TWFZGCMQGLPBSX-UHFFFAOYSA-N 0.000 description 1
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- 230000015572 biosynthetic process Effects 0.000 description 1
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- 210000004369 blood Anatomy 0.000 description 1
- JNPZQRQPIHJYNM-UHFFFAOYSA-N carbendazim Chemical compound C1=C[CH]C2=NC(NC(=O)OC)=NC2=C1 JNPZQRQPIHJYNM-UHFFFAOYSA-N 0.000 description 1
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Classifications
-
- 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
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Developmental Biology & Embryology (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- Environmental Sciences (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention discloses a kind of South America astral oil rattan rapid propagation methods.The present invention is using the branch of South America astral oil rattan current year raw non-lignifying as explant, by explant surface sterilization, explant culture evoked callus, the differentiation and elongation of callus induction bud, strengthening seedling and rooting, acclimatization and transplants and etc. obtain regeneration plant a kind of method, whole process carries out under controlled condition, it is not subject to seasonal restrictions, substantially reduce the breeding cycle, increase breeding rate, to meet the needs of to South America astral oil rattan high quality seedling, the popularization of South America astral oil rattan breeding is accelerated to have important practical significance, the researchs such as plukenetia volubilis linneo improvement of genes provide a kind of efficiently feasible technological means after or.
Description
Technical field
The invention belongs to cell engineering technical field of tissue culture, and in particular to a kind of side that astral oil rattan in South America quickly breeds
Method.
Background technology
South America astral oil rattan (Plukenetia volubilis L.) also known as plukenetia volubilis linneo, print plus fruit, print plus peanut etc., print
Add language to claim Sacha Inchi, is Euphorbiaceae perennial evergreen woody climber oil crops, it is Andean to originate in South America
Tropical rain forest area.Plant current year plantation current year can blossom and bear fruit, and enter high yield period within 2~3 years, and the phase of abounding with is up to 10 years
More than.The oil squeezed from the astral oil rattan seed of South America contain higher unsaturated fatty acid (especially linolenic and linoleic),
VAAnd VE, to adjustment human body blood fat, prevention of cardiovascular disease, maintenance skin it is anti-aging the effects that it is apparent, be widely used in food, system
The fields such as medicine, health care and cosmetics are known as " plant docosapentaenoic acid ".In addition, South America astral oil rattan is alternatively arranged as area of conceding the land to forestry
Seeds are substituted, are a kind of economic oil crops of great exploitation and utilization and extention foreground.
The time that South America astral oil rattan introduces China is shorter, at present mainly the Yunnan west and south, Guizhou and Hainan partially
There is plantation in area.Its seedling breeding is mainly that seed is directly sowed, grafted and the modes such as cutting propagation, though seed sowing is in the short time
It is interior to can get a large amount of seedling, but because South America astral oil rattan is cross-pollinatd plant, it is unstable that seed sows inhereditary feature, it is difficult to retain
The merit of parent；Propagation by grafiting operation sequence is cumbersome, and technology requires height, is not easy to grasp；Cutting propagation then needs a large amount of branches
Item, and breeding potential is relatively low, take it is longer, this largely affect South America astral oil rattan scale, industrialization production and
The requirement of the marketization.And plant tissue culture technique has short breeding coefficient height, repoductive time, improvement plant quality, not by season
The advantages that section limitation, the problems such as South America astral oil rattan breeding coefficient is low, repoductive time is long can be efficiently solved.
Currently, it is experiment material that the research about South America astral oil rattan tissue cultures, which is detected in the aseptic seedling obtained with seed sowing,
Material, using the terminal bud of aseptic seedling as explant, by the induction of bud, be proliferated, take root and transplant etc. modes achieve the purpose that it is micro- numerous, it is numerous
It is undesirable to grow effect.The Chinese patent literature of Publication No. CN 104798684A discloses a kind of tissue training of South America astral oil rattan
Support quick-breeding method, using South America astral oil rattan stem-segment with node as explant, by adventitious bud inducing, be proliferated, take root, acclimatization and transplants waited
South America astral oil rattan Regeneration in Vitro plant has successfully been obtained in journey.It is planted though this method successfully obtains South America astral oil rattan Regeneration in Vitro
Strain, but the inductivity of its adventitious bud is not high (92.7%), induction and the differentiation etc. for also not carrying out callus to explant are ground
Study carefully.
Invention content
It is an object of the present invention to provide a kind of methods of South America astral oil rattan nursery.
The method of astral oil rattan nursery in South America provided by the invention includes the following steps：
(1) explant of South America astral oil rattan on callus inducing medium is subjected to Fiber differentiation, obtains callus group
It knits；
(2) callus is carried out to induction differentiation culture on callus induction differential medium, it is indefinite to obtain
Bud；
(3) adventitious bud is carried out to strengthening seedling and rooting culture on strengthening seedling and rooting culture medium, obtains South America astral oil rattan seedling.
In the above method,
The explant of the South America astral oil rattan is South America astral oil rattan branch；The South America astral oil rattan branch is that current year is not raw wooden
The South America astral oil rattan branch of matter；
Solute in the callus inducing medium includes IBA, 6-BA and TDZ；
Solute in the callus induction differential medium includes IBA, CCC and TDZ；
Solute in the strengthening seedling and rooting culture medium includes IBA and AC；
The mass ratio of IBA, 6-BA and TDZ in the callus inducing medium are 1：(1-10)：(1-10)；
The mass ratio of IBA, CCC and TDZ in the callus induction differential medium are 1：(1-3)：(1-10)；
The mass ratio of IBA and AC in the strengthening seedling and rooting culture medium are (0.1-1)：1.
In the above method,
The callus inducing medium is to obtain basal medium, IBA, 6-BA, TDZ, sucrose and coagulator mixing
The culture medium arrived；
The callus induction differential medium is by basal medium, IBA, CCC, TDZ, sucrose and coagulator mixing
Obtained culture medium；
The strengthening seedling and rooting culture medium is the culture medium for being uniformly mixed so as to obtain basal medium, IBA, AC, sucrose and coagulator.
In the above method,
Basal medium in the callus inducing medium and the callus induction differential medium is MS
Culture medium, formula are as follows：NH4NO3 1.65g/L,H3BO3 6.2mg/L,CaCl2 332.2mg/L,CoCl2﹒ 6H2O
0.025mg/L,CuSO4﹒ 5H2O 0.025mg/L, EDETATE SODIUM dihydrate 37.26mg/L, FeSO4﹒ 7H2O 27.8mg/L,
MgSO4 180.7mg/L,MnSO4﹒ H2O 16.9mg/L,Na2MoO4﹒ 2H2O 0.25mg/L,KI 0.83mg/L,KNO3 1.9g/
L,KH2PO4 170mg/L,ZnSO4﹒ 7H2O 8.6mg/L。
Basal medium in the strengthening seedling and rooting culture medium can be above-mentioned MS culture mediums, can also be MSvitCulture
Base, the MSvitThe formula of culture medium is as follows：NH4NO3 1.65g/L,H3BO3 6.2mg/L,CaCl2 332.2mg/L,
CoCl2﹒ 6H2O 0.025mg/L,CuSO4﹒ 5H2O 0.025mg/L, EDETATE SODIUM dihydrate 37.26mg/L, FeSO4﹒ 7H2O
27.8mg/L, glycine 2mg/L, MgSO4 180.7mg/L,MnSO4﹒ H2O 16.9mg/L,Na2MoO4﹒ 2H2O 0.25mg/L,
Inositol 100mg/L, niacin 0.5mg/L, KI 0.83mg/L, KNO3 1.9g/L,KH2PO4170mg/L, puridoxine hydrochloride
0.5mg/L, thiamine hydrochloride 0.1mg/L, ZnSO4﹒ 7H2O 8.6mg/L。
In the above method,
A concentration of 0-1.0mg/Ls of the IBA in the callus inducing medium；
A concentration of 0-1.0mg/Ls of the 6-BA in the callus inducing medium；
A concentration of 0.5-2.0mg/Ls of the TDZ in the callus inducing medium；
A concentration of 0-2.0mg/Ls of the IBA in the callus induction differential medium；
A concentration of 0-1.0mg/Ls of the CCC in the callus induction differential medium；
A concentration of 0-1.0mg/Ls of the TDZ in the callus induction differential medium；
A concentration of 0-2.0mg/Ls of the IBA in the strengthening seedling and rooting culture medium；
A concentration of 0-1.0mg/Ls of the AC in the strengthening seedling and rooting culture medium.
In the above method,
The coagulator is agar；
A concentration of 0.5mg/Ls of the IBA in the callus inducing medium；
A concentration of 1.0mg/Ls of the 6-BA in the callus inducing medium；
A concentration of 0.5mg/Ls of the TDZ in the callus inducing medium；
A concentration of 0.1mg/Ls of the IBA in the callus induction differential medium；
A concentration of 0.1mg/Ls of the CCC in the callus induction differential medium；
A concentration of 0.1mg/Ls of the TDZ in the callus induction differential medium；
Concentration of the sucrose in the callus inducing medium and the callus induction differential medium
It is 30g/L；
The agar is in the callus inducing medium, the callus induction differential medium and the strong sprout
Concentration in root media is 8g/L；
A concentration of 0.1mg/Ls of the IBA in the strengthening seedling and rooting culture medium；
A concentration of 1g/Ls of the AC in the strengthening seedling and rooting culture medium；
A concentration of 40g/L of the sucrose in the strengthening seedling and rooting culture medium.
In the above method,
The pH of the culture medium is 5.8-6.0.
In the above method,
The condition of the culture is：23-27 DEG C of temperature, relative humidity 55-60%, 40 μm of ol m of intensity of illumination-2·s-1, light application time 12h/d.
In the above method,
It is described on callus inducing medium carry out induction of callus before further include the steps that disinfection；
Further include the steps that acclimatization and transplants after the strengthening seedling and rooting culture.
It is a further object to provide above-mentioned callus inducing mediums or above-mentioned callus induction to break up
Culture medium or above-mentioned strengthening seedling and rooting culture medium.
It is a still further object of the present invention to provide a kind of kits for South America astral oil rattan nursery.
Kit provided by the present invention for South America astral oil rattan nursery include above-mentioned callus inducing medium and/or
Above-mentioned callus induction differential medium and/or above-mentioned strengthening seedling and rooting culture medium.
Final object of the present invention is to provide the above method or above-mentioned callus inducing medium or above-mentioned callus
Organize the new application of inductive differentiation medium or above-mentioned strengthening seedling and rooting culture medium or mentioned reagent box.
The present invention provides the above method or the differentiation trainings of above-mentioned callus inducing medium or above-mentioned callus induction
Application during foster base or above-mentioned strengthening seedling and rooting culture medium or mentioned reagent box are any in following (1)-(4)：
(1) callus induction rate of South America astral oil rattan is improved；
(2) the adventitious bud number that South America astral oil rattan callus differentiates is improved；
(3) number of taking root of South America astral oil rattan adventitious bud is improved；
(4) survival rate of plant of South America astral oil rattan is improved.
The present invention's has the prominent advantages that：
(1) present invention to South America astral oil rattan carry out callus induction and differentiation test, by choose robust growth, when
The South America astral oil rattan branch of Nian Shengwei lignifyings is explant, carries out the induction differentiation of callus, cultivates in a short time
The excellent South America astral oil rattan seedling of a large amount of growing ways, Technical Reference is provided for its factorial praluction.
(2) the present invention relates to the plant hormones such as IBA, CCC, TDZ and 6-BA be remarkably improved callus inductivity and
It is differentiated to form the ability of adventitious bud；Wherein, TDZ (Thidiazuron) energy rapid induction explant is from calli induction to adventitious bud
Differentiation, AC etc. can inhibit the browning of explant and callus in incubation.
(3) using cultural method culture 20d of the present invention or so callus induction rate up to 100%, generation is cured
Injured tissue is in light green color, and quality is more loose, health；Continue 15~20d of culture, about 98% or more callus can induce differentiation shape
At adventitious bud, and the mean germination number of adventitious bud, up to 12.83, growing way is preferable.
The present invention provides one kind by obtaining a large amount of good South America astral oil rattans in the method for plant tissue culture short time
The method of seedling.Using the branch of South America astral oil rattan current year raw non-lignifying as explant, by explant surface sterilization, explant
Cultivate evoked callus, callus induction bud differentiation and elongation, strengthening seedling and rooting, acclimatization and transplants and etc. obtain regeneration plant
A kind of method of strain, whole process are carried out under controlled condition, are not subject to seasonal restrictions, and also substantially reduce the breeding cycle, are increased
Breeding rate, to meet, to South America astral oil rattan high quality seedling the needs of, to accelerate the popularization of South America astral oil rattan breeding to have important
Realistic meaning.In addition, most of genetic transforming method, which is all built upon cell and tissue, can regenerate intact plant
On basis, this method using the branch of South America astral oil rattan current year raw non-lignifying as explant, carry out callus induction and
Differentiation obtains regeneration plant, thus can filter out high yield, high-quality, disease-resistant, the stronger elite plant of resistance, can become later fast
Speed obtains a kind of efficiently feasible technological means of a large amount of high quality seedlings and improvement of genes.It is experimentally confirmed：The side of the present invention
Method can greatly speed up its proliferative speed, increase breeding rate, shorten the breeding time limit, for being widely applied for later South America astral oil rattan
Theoretical foundation and Technical Reference are provided with large-scale planting, while can also be improvement of genes, the heredity turn of later South America astral oil rattan
The researchs such as change lay the foundation.
Description of the drawings
Fig. 1 is explant culture evoked callus design sketch.
Fig. 2 is that callus induction is differentiated to form adventitious bud design sketch.
Fig. 3 is the elongation design sketch for being induced to differentiate to form bud.
Fig. 4 is strengthening seedling and rooting culture effect figure.
Fig. 5 is acclimatization and transplants design sketch.
Specific implementation mode
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
Quantitative test in following embodiments, is respectively provided with three repeated experiments, and results are averaged.
MS is used in callus inducing medium and callus induction differential medium in following embodiments
The bases Murashige＆Skoog salt is purchased from Hainan bio tech ltd Wei Ke, catalog number MSP01-50LT, tool
Body formula is as follows：NH4NO3 1.65g/L,H3BO3 6.2mg/L,CaCl2 332.2mg/L,CoCl2﹒ 6H2O 0.025mg/L,
CuSO4﹒ 5H2O 0.025mg/L, EDETATE SODIUM dihydrate 37.26mg/L, FeSO4﹒ 7H2O 27.8mg/L,MgSO4
180.7mg/L,MnSO4﹒ H2O 16.9mg/L,Na2MoO4﹒ 2H2O 0.25mg/L,KI 0.83mg/L,KNO3 1.9g/L,
KH2PO4 170mg/L,ZnSO4﹒ 7H2O 8.6mg/L。
MS used in strengthening seedling and rooting culture medium in following embodimentsvitTo contain vitaminic Murashige＆
The bases Skoog salt, purchased from Hainan bio tech ltd Wei Ke, catalog number MSP09-50LT, specific formula is as follows：
NH4NO3 1.65g/L,H3BO3 6.2mg/L,CaCl2 332.2mg/L,CoCl2﹒ 6H2O 0.025mg/L,CuSO4﹒ 5H2O
0.025mg/L, EDETATE SODIUM dihydrate 37.26mg/L, FeSO4﹒ 7H2O 27.8mg/L, glycine 2mg/L, MgSO4
180.7mg/L,MnSO4﹒ H2O 16.9mg/L,Na2MoO4﹒ 2H2O 0.25mg/L, inositol 100mg/L, niacin 0.5mg/L, KI
0.83mg/L,KNO3 1.9g/L,KH2PO4170mg/L, puridoxine hydrochloride 0.5mg/L, thiamine hydrochloride 0.1mg/L, ZnSO4﹒
7H2O 8.6mg/L。
IBA (indolebutyric acid) in following embodiments, is purchased in Jinan Prandtl bio tech ltd, catalogue
Number：PLT-02.
6-BA (6- benzyls aminoadenine) in following embodiments, is purchased in Jinan Prandtl bio tech ltd,
Catalog number：PLT-02.
TDZ (Thidiazuron) in following embodiments, is purchased in Jinan Prandtl bio tech ltd, catalogue
Number：51707-55-2.
CCC (cycocel) in following embodiments, is purchased in Jinan Prandtl bio tech ltd, catalogue
Number：724C032.
AC (ascorbic acid) in following embodiments, is purchased in Jinan Prandtl bio tech ltd, catalogue
Number：20150501-1.
NAA (a- methyl α-naphthyl acetates) in following embodiments, is purchased in Sinopharm Chemical Reagent Co., Ltd., catalogue
Number：F 20110613.
Embodiment 1, a kind of method of South America astral oil rattan nursery
One, explant acquisition and surface sterilization
The South America astral oil rattan branch that current year of clip robust growth gives birth to non-lignifying is taken back in sealed bag, and by acquisition
Fresh braches carry out surface sterilization.It is as follows：
1, branch is cut into the about stem section of 1.5cm long, be placed in 200ml beakers, first (carving board is washed clean with appropriate abluent
Agent is purchased from Carrefour hypermarket；Catalog number GB9985-2000) 30min is impregnated, beaker during which is jiggled per 3-5min, is soaked
30min is rinsed after bubble under flowing water to be placed on superclean bench, the explant after being impregnated；
2,30s is sterilized to the explant after immersion with 70% ethyl alcohol, with sterile water wash 5 times；It is sterilized again with 2%NaCl
8min, sterile water wash 5 times, aseptic filter paper blots the droplet on surface, the explant that is sterilized that treated, spare.
Two, explant culture evoked callus
With scalpel by step 1 obtain disinfect after the stem section both ends of explant cut away, according to molten in culture medium
Matter concentration difference be inoculated into respectively it is following a), b) and c) shown in callus inducing medium and callus induction control
Induction of callus is carried out on culture medium：
A) MS+0.1mg/L IBA+0.5mg/L 6-BA+0.5mg/L TDZ+30g/L sucrose+8g/L agar (pH=
5.8)。
B) MS+0.5mg/L IBA+1.0mg/L 6-BA+0.5mg/L TDZ+30g/L sucrose+8g/L agar (pH=
5.8)。
C) MS+1.0mg/L IBA+0.5mg/L 6-BA+1.0mg/L TDZ+30g/L sucrose+8g/L agar (pH=
5.8)。
Callus induction control medium：MS+30g/L sucrose+8g/L agar (pH=5.8).
Condition of culture：25 ± 2 DEG C of temperature, 40 μm of ol m of intensity of illumination-2·s-1, light application time 12h/d.
After explant culture 30 days, in addition to control medium, all culture mediums can pass through explant induced synthesis callus
Tissue, the difference is that the inductivity of callus and porousness difference.Explant is in each callus inducing medium
The result of Fiber differentiation is specific as follows：
Chartreuse callus is formed when being cultivated 7-10 days in culture medium shown in a), continues culture 15-25 days, callus
Tissue expands, light green color, more loose, and inductivity is up to 100%.
Chartreuse or light green color callus are formed when being cultivated 7-10 days in culture medium shown in b), continue to cultivate 15-
20 days, callus expanded, and light green color is more loose, and health, inductivity is up to 100%.B) Fiber differentiation in culture medium shown in
Explant is as shown in Figure 1.
Chartreuse callus is formed when being cultivated 7-10 days in culture medium shown in c), continues culture 15-30 days, callus
Tissue expands, loose, and the different degrees of browning of part callus, and inductivity is up to 95%.
By above-mentioned cultivation results it is found that different callus inducing mediums imitate the induction of South America astral oil rattan callus
Fruit is different, and difference is more significant.South America astral oil rattan callus inducing medium formula be preferably b) shown in culture medium (MS+
0.5mg/L IBA+1.0mg/L 6-BA+0.5mg/L TDZ+30g/L sucrose+8g/L agar, pH=5.8), what induction generated
Callus is more loose, healthy, and inductivity is up to 100%.
Three, the differentiation and elongation of callus induction bud
By in step 2 b) shown in culture medium Fiber differentiation obtain callus according to solute concentration in culture medium
Difference be inoculated into respectively it is following d), e) and f) shown in callus induction differential medium and callus induction differentiation control
Callus differentiation culture is carried out on culture medium：
D) MS+0.1mg/L IBA+0.1mg/L CCC+0.1mg/L TDZ+30g/L sucrose+8g/L agar (pH=5.8).
E) MS+0.5mg/L IBA+0.2mg/L CCC+1.0mg/L TDZ+30g/L sucrose+8g/L agar (pH=5.8).
F) MS+1.0mg/L IBA+0.3mg/L CCC+0.5mg/L TDZ+30g/L sucrose+8g/L agar (pH=5.8).
Callus induction breaks up control medium：MS+30g/L sucrose+8g/L agar (pH=5.8).
Condition of culture：25 ± 2 DEG C of temperature, 40 μm of ol m of intensity of illumination-2·s-1, light application time 12h/d.
After explant culture 45 days, the result of explant Fiber differentiation in each callus induction differential medium has
Body is as follows：
D) it cultivates 15-20 days, about 98% or more callus differentiates adventitious bud, after continuing culture 45 days, induction point
It is preferable to change the adventitious bud growing way generated, mean germination number is 12.83, a length of 6.64mm of average bud.D) in culture medium shown in
It is as shown in Figures 2 and 3 that callus differentiates adventitious bud.
E) it cultivates 15-30 days, about 90% or more callus is differentiated to form adventitious bud, average after continuing culture 45 days
Germinative number is 9.33, a length of 12.34mm of average bud.
F) it cultivates 15-30 days, about 70% or more callus is differentiated to form adventitious bud, after continuing culture 45 days, small portion
Callus browning, a little bud is divided to wilt, growing way is general, and mean germination number is 7.67, a length of 5.74mm of average bud.
Break up in control medium the generation for not finding callus in callus induction.
By above-mentioned cultivation results it is found that South America astral oil rattan callus induction differential medium formula be preferably d) shown in
It is lured culture medium (MS+0.1mg/L IBA+0.1mg/L CCC+0.1mg/L TDZ+30g/L sucrose+8g/L agar, pH=5.8)
Lead that differentiation budding growing way is more healthy, and bud number is most, up to 12.83, far above explant in 104798684 A of patent No. CN
The adventitious bud number being induced to differentiate to form.
Four, strengthening seedling and rooting culture
By in step 3 d) shown under the budlet cutting of height about 2.5-4cm that obtains of culture medium induction differentiation, and press
According to solute in culture medium and its concentration difference be inoculated into respectively g), h) and i) shown in carry out strong sprout on strengthening seedling and rooting culture medium
Culture of rootage：
g)MS vit+ 0.1mg/L IBA+1g/L AC+40g/L sucrose+8g/L agar.
h)MS vit+ 0.5mg/L IBA+0.5mg/L NAA+0.1mg/L 6-BA+1g/L AC+40g/L sucrose+8g/L fine jades
Fat.
i)MS vit+ 1.0mg/L IBA+1.0mg/L NAA+0.5mg/L 6-BA+1g/L AC+40g/L sucrose+8g/L fine jades
Fat.
Condition of culture is：25 ± 2 DEG C of temperature, 40 μm of ol m of intensity of illumination-2·s-1, light application time 12h/d.
After culture 60 days, the result that each budlet is cultivated in each strengthening seedling and rooting culture medium is specific as follows：
G) it cultivates 15 days or so and starts to take root, rooting rate 100% after continuing culture 60 days, is averagely taken root and counted up to 10.6
Item, a length of 85.6mm of average root, growing way are preferable.G) seedling of adventitious bud is as shown in Figure 4 in culture medium shown in.
H) it cultivates 30 days or so and starts long root, after continuing culture 60 days, negligible amounts of taking root, but root is more sturdy.
I) there is a little long root after cultivating 60 days, rooting efficiency is poor, the gradual yellow of bud-leaf piece.
By above-mentioned cultivation results it is found that South America astral oil rattan strengthening seedling and rooting culture medium prescription be preferably g) shown in culture medium
(MS vit+ 0.1mg/L IBA+1g/L AC+40g/L sucrose+8g/L agar, pH=5.8), induce the number of taking root that is averaged generated
More, average root long is longer, and plant growing way is preferable.
Five, acclimatization and transplants
By in step 4 g) shown in medium culture obtain high about 4-6cm, robust growth, root system is sturdy, leaf color is dense
Green rooting tube plantlet is placed in greenhouse, and temperature is maintained at 25 DEG C or so, opens bottle cap after natural lighting lower refining seedling 15 days, will
Test tube seedling is taken out from culture bottle, washes off root culture medium and with 10s is impregnated in carbendazim solution, is transplanted to the cultivation of disinfection
Training matrix, (proportioning of cultivation matrix is peat soil：Coconut palm chaff：Perlite=3:2:1) culture obtains regeneration plant in, periodically waters,
Holding light transmittance is 70-80%, 80% or more relative humidity.Regeneration plant survival rate is up to 85% or more, regeneration plant such as Fig. 5 institutes
Show.
Claims (6)
1. a kind of method of South America astral oil rattan nursery, includes the following steps：
(1) explant of South America astral oil rattan on callus inducing medium is subjected to Fiber differentiation, obtains callus；
(2) callus is carried out to induction differentiation culture on callus induction differential medium, obtains adventitious bud；
(3) adventitious bud is carried out to strengthening seedling and rooting culture on strengthening seedling and rooting culture medium, obtains South America astral oil rattan seedling；
The explant of the South America astral oil rattan is South America astral oil rattan branch；The South America astral oil rattan branch is current year raw non-lignifying
South America astral oil rattan branch；
Solute in the callus inducing medium includes IBA, 6-BA and TDZ；
Solute in the callus induction differential medium includes IBA, CCC and TDZ；
Solute in the strengthening seedling and rooting culture medium includes IBA and AC；
A concentration of 0.1-1.0mg/Ls of the IBA in the callus inducing medium；
A concentration of 0.5-1.0mg/Ls of the 6-BA in the callus inducing medium；
A concentration of 0.5-1.0mg/Ls of the TDZ in the callus inducing medium；
A concentration of 0.1-1.0mg/Ls of the IBA in the callus induction differential medium；
A concentration of 0.1-0.3mg/Ls of the CCC in the callus induction differential medium；
A concentration of 0.1-1.0mg/Ls of the TDZ in the callus induction differential medium；
A concentration of 0.1-0.5mg/Ls of the IBA in the strengthening seedling and rooting culture medium；
A concentration of 1.0g/Ls of the AC in the strengthening seedling and rooting culture medium.
2. according to the method described in claim 1, it is characterized in that：
Basal medium, IBA, 6-BA, TDZ, sucrose and coagulator are uniformly mixed so as to obtain by the callus inducing medium
Culture medium；
The callus induction differential medium is to be uniformly mixed so as to obtain basal medium, IBA, CCC, TDZ, sucrose and coagulator
Culture medium；
The strengthening seedling and rooting culture medium is the culture medium for being uniformly mixed so as to obtain basal medium, IBA, AC, sucrose and coagulator.
3. according to the method described in claim 2, it is characterized in that：
The coagulator is agar；
A concentration of 0.5mg/Ls of the IBA in the callus inducing medium；
A concentration of 1.0mg/Ls of the 6-BA in the callus inducing medium；
A concentration of 0.5mg/Ls of the TDZ in the callus inducing medium；
A concentration of 0.1mg/Ls of the IBA in the callus induction differential medium；
A concentration of 0.1mg/Ls of the CCC in the callus induction differential medium；
A concentration of 0.1mg/Ls of the TDZ in the callus induction differential medium；
Concentration of the sucrose in the callus inducing medium and the callus induction differential medium is
30g/L；
The agar is in the callus inducing medium, the callus induction differential medium and the strengthening seedling and rooting
Concentration in culture medium is 8g/L；
A concentration of 0.1mg/Ls of the IBA in the strengthening seedling and rooting culture medium；
A concentration of 1g/Ls of the AC in the strengthening seedling and rooting culture medium；
A concentration of 40g/L of the sucrose in the strengthening seedling and rooting culture medium.
4. according to any method in claim 1-3, it is characterised in that：
The pH of the culture medium is 5.8-6.0；
The condition of the culture is：23-27 DEG C of temperature, relative humidity 55-60%, 40 μm of ol m of intensity of illumination-2·s-1, illumination
Time 12h/d.
5. according to any method in claim 1-3, it is characterised in that：
It is described on callus inducing medium carry out induction of callus before further include the steps that disinfection；
Further include the steps that acclimatization and transplants after the strengthening seedling and rooting culture.
The application during 6. any methods of claim 1-5 are any in following (1)-(4)：
(1) callus induction rate of South America astral oil rattan is improved；
(2) the adventitious bud number that South America astral oil rattan callus differentiates is improved；
(3) number of taking root of South America astral oil rattan adventitious bud is improved；
(4) survival rate of plant of South America astral oil rattan is improved.
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| CN114503918A (en) * | 2022-03-21 | 2022-05-17 | 玉林师范学院 | Method for promoting proliferation of adventitious buds of plukenetia volubilis linneo |
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