CN101897298B - Method for inducing eucalyptus to generate calli and differentiating calli into buds - Google Patents
Method for inducing eucalyptus to generate calli and differentiating calli into buds Download PDFInfo
- Publication number
- CN101897298B CN101897298B CN 201010249030 CN201010249030A CN101897298B CN 101897298 B CN101897298 B CN 101897298B CN 201010249030 CN201010249030 CN 201010249030 CN 201010249030 A CN201010249030 A CN 201010249030A CN 101897298 B CN101897298 B CN 101897298B
- Authority
- CN
- China
- Prior art keywords
- medium
- bud
- callus
- days
- illumination
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
- 238000000034 method Methods 0.000 title claims abstract description 24
- 230000001939 inductive effect Effects 0.000 title claims abstract description 17
- 244000166124 Eucalyptus globulus Species 0.000 title claims abstract 9
- 206010020649 Hyperkeratosis Diseases 0.000 claims abstract description 51
- 230000006698 induction Effects 0.000 claims abstract description 17
- 230000001954 sterilising effect Effects 0.000 claims abstract description 6
- 238000005286 illumination Methods 0.000 claims description 40
- 239000006870 ms-medium Substances 0.000 claims description 35
- 238000012546 transfer Methods 0.000 claims description 25
- KIDHWZJUCRJVML-UHFFFAOYSA-N putrescine Chemical compound NCCCCN KIDHWZJUCRJVML-UHFFFAOYSA-N 0.000 claims description 22
- ATHGHQPFGPMSJY-UHFFFAOYSA-N spermidine Chemical compound NCCCCNCCCN ATHGHQPFGPMSJY-UHFFFAOYSA-N 0.000 claims description 22
- 239000006160 differential media Substances 0.000 claims description 16
- 230000004069 differentiation Effects 0.000 claims description 16
- YGGXZTQSGNFKPJ-UHFFFAOYSA-N methyl 2-naphthalen-1-ylacetate Chemical compound C1=CC=C2C(CC(=O)OC)=CC=CC2=C1 YGGXZTQSGNFKPJ-UHFFFAOYSA-N 0.000 claims description 15
- PVRBGBGMDLPYKG-UHFFFAOYSA-N 6-benzyl-7h-purine Chemical compound N=1C=NC=2N=CNC=2C=1CC1=CC=CC=C1 PVRBGBGMDLPYKG-UHFFFAOYSA-N 0.000 claims description 14
- 239000002609 medium Substances 0.000 claims description 12
- 239000005700 Putrescine Substances 0.000 claims description 11
- 229940063673 spermidine Drugs 0.000 claims description 11
- 238000005406 washing Methods 0.000 claims description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 11
- 229930006000 Sucrose Natural products 0.000 claims description 8
- 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 8
- 238000004659 sterilization and disinfection Methods 0.000 claims description 8
- 239000005720 sucrose Substances 0.000 claims description 8
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 7
- 229960002523 mercuric chloride Drugs 0.000 claims description 6
- LWJROJCJINYWOX-UHFFFAOYSA-L mercury dichloride Chemical compound Cl[Hg]Cl LWJROJCJINYWOX-UHFFFAOYSA-L 0.000 claims description 6
- 239000004202 carbamide Substances 0.000 claims description 5
- JTEDVYBZBROSJT-UHFFFAOYSA-N indole-3-butyric acid Chemical compound C1=CC=C2C(CCCC(=O)O)=CNC2=C1 JTEDVYBZBROSJT-UHFFFAOYSA-N 0.000 claims description 5
- 239000002253 acid Substances 0.000 claims description 4
- 230000004913 activation Effects 0.000 claims description 4
- 230000020411 cell activation Effects 0.000 claims description 3
- 244000080540 Eucalyptus hybrid Species 0.000 claims description 2
- 235000006913 Eucalyptus hybrid Nutrition 0.000 claims description 2
- 230000008929 regeneration Effects 0.000 abstract description 20
- 238000011069 regeneration method Methods 0.000 abstract description 20
- 108090000623 proteins and genes Proteins 0.000 abstract description 9
- 230000001172 regenerating effect Effects 0.000 abstract description 4
- 230000002068 genetic effect Effects 0.000 abstract description 3
- 230000006872 improvement Effects 0.000 abstract description 3
- 238000010353 genetic engineering Methods 0.000 abstract description 2
- 239000004952 Polyamide Substances 0.000 abstract 1
- 239000001963 growth medium Substances 0.000 abstract 1
- 229920002647 polyamide Polymers 0.000 abstract 1
- 241000219927 Eucalyptus Species 0.000 description 31
- WLJVXDMOQOGPHL-UHFFFAOYSA-N phenylacetic acid Chemical compound OC(=O)CC1=CC=CC=C1 WLJVXDMOQOGPHL-UHFFFAOYSA-N 0.000 description 12
- 102000018997 Growth Hormone Human genes 0.000 description 9
- 108010051696 Growth Hormone Proteins 0.000 description 9
- 238000009395 breeding Methods 0.000 description 9
- 239000000122 growth hormone Substances 0.000 description 9
- 230000001488 breeding effect Effects 0.000 description 8
- 241000196324 Embryophyta Species 0.000 description 7
- 230000032823 cell division Effects 0.000 description 7
- 229960003424 phenylacetic acid Drugs 0.000 description 6
- 239000003279 phenylacetic acid Substances 0.000 description 6
- 230000032459 dedifferentiation Effects 0.000 description 5
- 238000013459 approach Methods 0.000 description 4
- RCTYPNKXASFOBE-UHFFFAOYSA-M chloromercury Chemical compound [Hg]Cl RCTYPNKXASFOBE-UHFFFAOYSA-M 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 229920000768 polyamine Polymers 0.000 description 4
- 241000006109 Eucalyptus delegatensis Species 0.000 description 3
- 210000004027 cell Anatomy 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 210000001082 somatic cell Anatomy 0.000 description 3
- 230000009261 transgenic effect Effects 0.000 description 3
- 241000404037 Eucalyptus urophylla Species 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 230000008676 import Effects 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 230000009466 transformation Effects 0.000 description 2
- OVSKIKFHRZPJSS-UHFFFAOYSA-N 2,4-D Chemical compound OC(=O)COC1=CC=C(Cl)C=C1Cl OVSKIKFHRZPJSS-UHFFFAOYSA-N 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 244000165963 Eucalyptus camaldulensis Species 0.000 description 1
- 241001256872 Perula Species 0.000 description 1
- 240000005860 Portulaca grandiflora Species 0.000 description 1
- 229920001131 Pulp (paper) Polymers 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000009402 cross-breeding Methods 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 230000014509 gene expression Effects 0.000 description 1
- 238000012239 gene modification Methods 0.000 description 1
- 230000005017 genetic modification Effects 0.000 description 1
- 235000013617 genetically modified food Nutrition 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 238000007654 immersion Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000001823 molecular biology technique Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000000392 somatic effect Effects 0.000 description 1
- 239000001648 tannin Substances 0.000 description 1
- 229920001864 tannin Polymers 0.000 description 1
- 235000018553 tannin Nutrition 0.000 description 1
- 238000012090 tissue culture technique Methods 0.000 description 1
- 239000000341 volatile oil Substances 0.000 description 1
Landscapes
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention discloses a method for inducing a eucalyptus to generate calli and differentiating the calli into buds, which comprises the following steps of: collecting auxiliary buds, sterilizing the auxiliary buds and allowing the auxiliary buds to spout; performing root induction and albino seedling induction cultivation; performing induction cultivation by using cut stem segments of albino seedlings as explants to obtain the calli, wherein a callus induction culture medium contains polyamides; and differentiating the generated calli to obtain adventitious buds. In the method, the buds are differentiated by the regeneration of the stem segment calli of the albino seedlings, and the callus regeneration can be used in genetic engineering, in which target genes are transferred into recipient cells, and the like and is the basis of transgenosis. Moreover, based on the budling of clonal adult eucalyptuses growing in a tree farm, the regenerating system has a regenerating efficiency of over 30 percent and can be applied to DH32-29, Guang tree 9 and other excellent clones. Thus, the method provides a good basis for the genetic improvement on the eucalyptus by using modern biotechnology and has a very important economical value.
Description
Technical field
The present invention relates to the territory, tissue culture technique field of eucalyptus, refer in particular to a kind of inducing eucalyptus and produce callus and break up the method for sprouting.
Background technology
Eucalyptus is the general designation that Myrtaceae Myrtaceae eucalyptus belongs to the Eucalyptus plant.Most eucalyptus are evergreen high megaphanerophyte, and minority is dungarunga or shrub.Eucalyptus is distributed in the torrid zone and subtropical zone, belongs to sun plant, and the sturdy prosperity of root system.Eucalyptus has fast-growing, high yield, and strong adaptability, with short production cycle, the characteristics such as purposes is wide are one of main quick-growing cultivation seeds that can be used as paper pulp material.Eucalyptus is extensively planted by a plurality of countries and regions at present, and the eucalyptus plantation area accounts for 1/3rd of world's forest plantation gross area.Eucalyptus can also as the raw material that extracts tannin and essential oil, therefore have widely value of exploiting and utilizing except the purposes as pulpwood and fibrous plate.
Through the research of organizing the clones such as cultivation and cuttage to grow seedlings for many years, and crossbreeding and select excellent traditional seed selection breeding work, producing at present upward widely used is exactly the Hybrid of alpine ash and Eucalyptus urophylla, for example DH32-29, extensively woods No. nine and other choiceness.
Because the tree growth cycle is long, heredity is assorted and property is high, and many proterties belong to the quantitative character of controlled by multiple genes, and conventional breeding technique is difficult to satisfy the requirement of directive breeding eucalyptus new varieties.Therefore people wish to utilize emerging technique for gene engineering to solve the insoluble problem of eucalyptus conventional breeding, directionally cultivate the high proterties of economic worth.Genetic engineering breeding is take modern molecular biology technique as the basis, and genes of interest is imported recipient cell by vitro recombination, and then bears whole plant with make new advances a kind of technological means of eucalypt species of cultivation.It has high efficiency and specific aim, can remedy the deficiency of traditional breeding method, accelerates high-quality, the seed selection of high value eucalyptus new varieties.
Utilize at present plant transgenic technology to have several problems: the one, transformation efficiency is on the low side; The 2nd, main transformation technology is limited to the kinds such as eucalyptus camaldulensis, alpine ash, and report seldom in other eucalypt specieses; The 3rd, the experiment material that most of success is regenerated is that seed variation is large, is difficult to be applied to clonal eucalyptus crossbreed take seed as the basis.Because extensively the choiceness such as No. nine, the DH32-29 of cultivation, wide woods are extremely low by the regeneration efficiency of leaf dish and callus from stem segment, thereby the independent transformed plant that is difficult to obtain a greater number filters out the good strain of exogenous gene expression, and greatly restriction is received in the development of transgenic breeding.
Summary of the invention
Technical problem to be solved by this invention is: the method that provides a kind of inducing eucalyptus to produce callus and differentiate, solve the low conversion rate that existing eucalyptus callus differentiation is sprouted, and can not carry out the callus differentiation to crossbreed and sprout, thus the problems such as restriction gene breeding.
For solving the problems of the technologies described above, the present invention adopts following technical scheme: a kind of inducing eucalyptus produces callus and breaks up the method for sprouting, and may further comprise the steps:
1) gathers axillalry bud, carry out disinfection and rudiment;
2) root induction and Albino Seedling are induced cultivation;
3) induce cultivation take the stem section segment of Albino Seedling as explant and obtain callus, this calli induction media contains polyamines;
4) Calli Differentiation that produces is gone out indefinite bud.
Described the 1st step is the stem segment with axillary bud that gathers the livings eucalyptus Hybrid of 2-4, is seeded in that sprouting obtains aseptic bud on the MS medium after sterile-processed; The condition of culture of rudiment is: temperature 22-30 ℃, and unglazed photograph; The method of described sterilization is to adopt alcohol and mercuric chloride sterilization, and uses aseptic water washing.
The 2nd step specifically may further comprise the steps:
A) (1) step is obtained the axillary bud sprouting bud and transfer to and contain the 0.1-2mg/L basic element of cell division, induce the generation Multiple Buds on the MS medium of 0.01-1mg/L growth hormone;
B) the healthy and strong bud clump in the Multiple Buds is transferred on the 1/2MS medium that contains 0.1-1mg/L growth hormone and induces the seedling of taking root;
C) seedling of will taking root cuts away terminal bud, is seeded in to contain sucrose 30-100g/L, NH
4NO
31650-6000mg/L, KH
2PO
4On the MS medium of 170-800mg/L, carry out in the dark Albino Seedling and induce cultivation.
Preferably, the 2nd step specifically comprises:
A) cut the axillalry bud of certain-length, transfer to and contain 0.1-2mg/L 6-benzyl purine, on the MS medium of 0.01-1mg/L methyl α-naphthyl acetate, cultivated 21-28 days, illumination 12-18 hour/day, intensity of illumination 1500-10000lx, temperature 22-30 ℃;
B) cut the bud that grows to certain-length, transfer to and carried out on the 1/2MS medium that contains 0.1-1mg/L 3-indolebutyric acid root induction 15-30 days, illumination 12-18 hour/day, intensity of illumination 1500-10000lx, temperature 22-30 ℃;
C) seedling of will taking root cuts away terminal bud, keeps several axillalry buds of root and base portion, is seeded in to contain sucrose 30-100g/L, NH
4NO
34500mg/L, KH
2PO
4Carry out Albino Seedling on the MS medium of 500mg/L and induce cultivation, secretly cultivated temperature 22-30 ℃ 14-35 days.
In the 3rd step, calli induction media preferably contains the MS medium of 50-200mg/L putrescine, 5-30mg/L spermidine.
In the 3rd step, calli induction media further comprises the 0.2-2mg/L basic element of cell division, 0.01-0.1mg/L growth hormone.
Preferably, the 3rd step specifically comprises:
A) take the stem section segment of Albino Seedling as explant, be seeded in and contain 0.05-0.3mg/L 2, the 4-dichlorphenoxyacetic acid carries out cell activation and cultivates on the MS medium of 0.1-0.5mg/L 6-benzyl purine, secretly cultivated temperature 22-30 ℃ 0-6 days;
B) explant after the activation is transferred to and is contained 0.2-2mg/L N-phenyl-N-1,2,3-thiadiazoles-5-urea, 0.01-0.1mg/L methyl α-naphthyl acetate, the 50-200mg/L putrescine is cultivated on the MS medium of 5-30mg/L spermidine and is produced callus, dark cultivation 5-7 days, temperature 22-30 ℃;
C) transfer to the low light level according to cultivation 12-14 days, illumination 12-18 hour/day, intensity of illumination 20-200lx, 22-30 ℃.
Described the 4th step is the callus that (3) step produces to be transferred to cultivate on the differential medium, makes the Calli Differentiation indefinite bud, and described differential medium is the MS medium that contains polyamines.
Wherein, the differential medium of the 4th step further comprises the 0.2-2mg/L basic element of cell division, 0.05-0.5mg/L growth hormone.
Preferably, described the 4th step specifically comprises:
A) with the callus that obtains, transfer to and contain 0.2-2mg/L 6-benzyl purine, 0.05-0.5mg/L methyl α-naphthyl acetate, the 10-40mg/L putrescine, 0.5-3mg/L on the MS medium of spermidine, transferred to subculture cultivation on the identical differential medium, illumination 12-18 hour/day every 14-25 days, intensity of illumination 500-3000lx, temperature 22-30 ℃;
B) subculture is cultivated and was produced indefinite bud in 14-100 days on differential medium.
Beneficial effect of the present invention is as follows: the present invention utilizes the stem section of Albino Seedling, sprouts through the differentiation of callus regeneration approach, and the callus regeneration approach can be used for genes of interest and import the gene engineerings such as recipient cell, is genetically modified basis.And, the inducing eucalyptus crossbreed that the present invention sets up produces callus and breaks up the method for sprouting, the grow up rudiment bar of eucalyptus of the clone of growing in the forest farm is the basis, the regeneration efficiency of regenerative system reaches more than 30%, can be applied to DH32-29, wide woods No. nine and other choiceness, for utilizing modern biotechnology that the genetic improvement of eucalyptus is had laid a good foundation, has very important economic worth.
Embodiment
The invention discloses a kind of inducing eucalyptus crossbreed and produce callus and break up the method for sprouting, may further comprise the steps:
(1) gathers axillalry bud, carry out disinfection and rudiment;
(2) root induction and Albino Seedling are induced cultivation;
(3) callus induces;
(4) differentiate indefinite bud.
In (1) step, gather the rudiment bar that 2-4 gives birth to hybridization eucalyptus new life, the preferred acquisition axillalry bud begins to expand, the perula sheet is uncracked rudiment bar still, be cut into the long stems with bud of 1.5-2.0cm, be seeded on the MS medium after sterile-processed and sprout, obtain aseptic bud.
The sterilization method of stem section is:
(a) 75% alcohol immersion is 20 seconds;
(b) aseptic water washing is 2 times;
(c) 0.1% mercuric chloride soaked 5 minutes;
(d) pour out mercuric chloride, use aseptic water washing 4-6 time;
(e) be trimmed to the long stems with bud of 0.5-1.0cm;
(f) 0.1% mercuric chloride soaked 2 minutes;
(g) pour out mercuric chloride, use aseptic water washing 4-6 time;
(h) material hidden that connects was cultivated 21-28 days.
The medium that rudiment is used is that the MS minimal medium contains 30g/L sucrose, 7g/L agar, and pH5.8.
The condition of culture of rudiment is, temperature 22-30 ℃, and unglazed photograph.
In (2) step, further comprise following incubation step:
(a) cut the long axillalry bud of 1.0cm, transfer to and contain the 0.1-2mg/L basic element of cell division such as 6-benzyl purine, on the MS medium of 0.01-1mg/L growth hormone such as methyl α-naphthyl acetate, cultivated 21-28 days, illumination 12-18 hour/day, intensity of illumination 1500-10000lx, temperature 22-30 ℃;
(b) cut the bud that grows to 1.5cm length, transfer to and carried out on the 1/2MS medium that contains 0.1-1mg/L growth hormone such as 3-indolebutyric acid root induction 15-30 days, illumination 12-18 hour/day, intensity of illumination 1500-10000lx, temperature 22-30 ℃;
(c) seedling of will taking root cuts away terminal bud, keeps 1-4 axillalry bud of root and base portion, is seeded in to contain sucrose 30-100g/L, NH
4NO
31650-6000mg/L, KH
2PO
4Carry out Albino Seedling on the MS medium of 170-800mg/L and induce cultivation, secretly cultivated 14-35 days, temperature 22-30 ℃, this MS inducing culture preferably contains NH
4NO
34500mg/L, KH
2PO
4500mg/L.
Wherein (3) step further comprises following steps:
(a) take the stem section segment of the 3-10mm of Albino Seedling as explant, be seeded in and contain 0.05-0.3mg/L growth hormone such as 2,4-dichlorphenoxyacetic acid, carry out cell activation on the MS medium of the 0.1-0.5mg/L basic element of cell division such as 6-benzyl purine and cultivate, dark cultivation 0-6 days, temperature 22-30 ℃;
(b) explant after the activation is transferred to and is contained the 0.2-2mg/L basic element of cell division such as N-phenyl-N-1,2,3-thiadiazoles-5-urea, 0.01-0.1mg/L growth hormone such as methyl α-naphthyl acetate, cultivate on the MS medium of polyamines such as 50-200mg/L putrescine, 5-30mg/L spermidine and produce callus, dark cultivation 5-7 days, temperature 22-30 ℃;
(c) transfer to the low light level according to cultivation 12-14 days, illumination 12-18 hour/day, intensity of illumination 20-200lx, 22-30 ℃.
In this step, with not cultivating with the stem section of the former base of bud of Albino Seedling, make the activated and dedifferentiation of the somatic cell of differentiation form callus.
And (4) step further comprises:
(a) with the callus that obtains, transfer to and contain the 0.2-2mg/L basic element of cell division such as 6-benzyl purine, 0.05-0.5mg/L growth hormone such as methyl α-naphthyl acetate, on the MS medium of polyamines such as 10-40mg/L putrescine, 0.5-3mg/L spermidine, transferred to subculture cultivation on the identical differential medium every 14-25 days.The condition of dedifferentiation culture is: illumination 12-18 hour/day, and intensity of illumination 500-3000lx, temperature 22-30 ℃;
(b) subculture is cultivated and was produced indefinite bud in 14-100 days on differential medium.
Method of the present invention is mainly utilized the stem section of Albino Seedling, sprouts through the differentiation of callus regeneration approach, and wherein the somatic cell that comprises by differentiation of Regeneration Ways forms callus through dedifferentiation, and then is differentiated to form bud.This callus can be used for genes of interest is imported recipient cell through Regeneration Ways, and then bear whole plant, thus can realize eucalyptus, particularly to the eucalyptus of Hybrid, such as DH32-29, wide woods No. nine and other choiceness, carry out directed genetic improvement.
Below in conjunction with example in detail description embodiment of the present invention are described in detail.Need to prove, following example is illustrative, is not determinate, can not limit protection scope of the present invention with following example.
Example 1: phenylacetic acid shown in the associative list 1, inducing eucalyptus stem section produces callus and breaks up the method for sprouting in this example, specifically may further comprise the steps:
At first, choose 3 years living plant of DH32-29, cut down the tree crown part, keep the high stub of 1-1.5m.Gather newborn rudiment bar after 1 month, be cut into the long stems with bud of 2.0cm, soaked 20 seconds in 70% ethanol, aseptic water washing 2 times, then sterilized 5 minutes in 0.1% mercury chloride, aseptic water washing 4 times is trimmed to the long stems with bud of 1.0cm, in 0.1% mercury chloride, sterilized 2 minutes again aseptic water washing 6 times.Be seeded in after drying in the MS medium, every bottle graft 1-2, secretly cultivated the axillalry bud that obtains sprouting 28 days for 25 ℃.
Second step cuts the long axillalry bud of 1.0cm, transfers to contain 0.1mg/L 6-benzyl purine, on the MS medium of 0.01mg/L methyl α-naphthyl acetate, transfers to subculture cultivation on the identical medium, illumination 16 hours/day, intensity of illumination 3000lx, 25 ℃ of temperature every 23 days.
The 3rd step cut and grows to the long bud of 1.5cm, transferred on the 1/2MS medium that contains 0.5mg/L 3-indolebutyric acid and carried out root induction 20 days, illumination 16 hours/day, intensity of illumination 3000lx, 25 ℃ of temperature.
In the 4th step, the seedling of will taking root cuts away terminal bud, keeps 2 axillalry buds of root and base portion, is seeded in to contain sucrose 70g/L, NH
4NO
34500mg/L, KH
2PO
4Carry out Albino Seedling on the MS medium of 500mg/L and induce cultivation, secretly cultivated 23 days, 25 ℃ of temperature, thus obtain Albino Seedling.
The 5th step, take the stem section segment of the 5mm of Albino Seedling as explant, totally 505 explants in this example, be seeded in and contain 0.1mg/L 2, the 4-dichlorphenoxyacetic acid is on the MS medium of 0.25mg/L6-benzyl purine, the dark cultivation 5 days, 25 ℃ of temperature, thereby activation somatic cell.
The 6th goes on foot, transfer to contain 0.7mg/L N-phenyl-N-1, and 2,3-thiadiazoles-5-urea, the 0.02mg/L methyl α-naphthyl acetate, the 80mg/L putrescine on the MS medium of 15mg/L spermidine, was secretly cultivated 5 days 25 ℃ of temperature.
The 7th step, transfer to the low light level according to cultivating 14 days, illumination 16 hours/day, intensity of illumination 50lx, 25 ℃ of temperature, thus obtain totally 505 of the callus of dedifferentiation.
The 8th step, with the callus that obtains, transfer to and contain 0.5mg/L 6-benzyl purine, 0.1mg/L methyl α-naphthyl acetate, the 15mg/L putrescine is on the MS medium of 1.5mg/L spermidine, transferred to subculture cultivation on the identical differential medium every 21 days, illumination 16 hours/day, intensity of illumination 1500lx, 25 ℃ of temperature.
At last, subculture is cultivated and was produced indefinite bud, totally 184 in 40 days on differential medium.
Specifically see Table 1:
Phenylacetic acid in table 1 example of the present invention
| Explant sum (piece) | Callus number (piece) | Differentiation callus number (piece) | Regeneration efficiency (%) |
| 505 | 505 | 184 | 36.4 |
In this example, the regeneration efficiency of callus regeneration system reaches 36.4%.
Example 2: phenylacetic acid shown in the associative list 2, inducing eucalyptus stem section produces callus and breaks up the method for sprouting in this example, specifically may further comprise the steps:
At first, choose 3 years living plant of wide woods No. nine, cut down the tree crown part, keep the high stub of 1-1.5m.Gather newborn rudiment bar after 1 month, be cut into the long stems with bud of 2.0cm, soaked 20 seconds in 70% ethanol, aseptic water washing 2 times, then sterilized 5 minutes in 0.1% mercury chloride, aseptic water washing 4 times is trimmed to the long stems with bud of 1.0cm, in 0.1% mercury chloride, sterilized 2 minutes again aseptic water washing 6 times.Be seeded in after drying in the MS medium, every bottle graft 1-2, secretly cultivated the axillalry bud that obtains sprouting 28 days for 25 ℃.
Second step cuts the long axillalry bud of 1.0cm, transfers to contain 0.1mg/L 6-benzyl purine, on the MS medium of 0.01mg/L methyl α-naphthyl acetate, transfers to subculture cultivation on the identical medium, illumination 16 hours/day, intensity of illumination 3000lx, 25 ℃ of temperature every 23 days.
The 3rd step cut and grows to the long bud of 1.5cm, transferred on the 1/2MS medium that contains 0.5mg/L 3-indolebutyric acid and carried out root induction 30 days, illumination 16 hours/day, intensity of illumination 3000lx, 25 ℃ of temperature.
In the 4th step, the seedling of will taking root cuts away terminal bud, keeps 2 axillalry buds of root and base portion, is seeded in to contain sucrose 50g/L, NH
4NO
34500mg/L, KH
2PO
4Carry out Albino Seedling on the MS medium of 500mg/L and induce cultivation, secretly cultivated 30 days, 25 ℃ of temperature.
The 5th step take the stem section segment of the 4mm of Albino Seedling as totally 321 of explants, was seeded in and contains 0.1mg/L 2, and the 4-dichlorphenoxyacetic acid on the MS medium of 0.25mg/L 6-benzyl purine, was secretly cultivated 6 days 25 ℃ of temperature.
The 6th goes on foot, transfer to contain 0.7mg/L N-phenyl-N-1, and 2,3-thiadiazoles-5-urea, the 0.02mg/L methyl α-naphthyl acetate, the 80mg/L putrescine on the MS medium of 15mg/L spermidine, was secretly cultivated 5 days 25 ℃ of temperature.
In the 7th step, transfer to the low light level according to cultivating illumination 16 hours/day, intensity of illumination 50lx, 25 ℃ of temperature 14 days.
The 8th step, with totally 321 of the callus that obtain, transfer to and contain 0.5mg/L 6-benzyl purine, 0.1mg/L methyl α-naphthyl acetate, the 15mg/L putrescine is on the MS medium of 1.5mg/L spermidine, transferred to subculture cultivation on the identical differential medium every 21 days, illumination 16 hours/day, intensity of illumination 1500lx, 25 ℃ of temperature.
At last, subculture is cultivated and was produced indefinite bud, totally 136 in 30 days on differential medium.
Specifically see Table 2:
Phenylacetic acid in table 2 example of the present invention
| Explant sum (piece) | Callus number (piece) | Differentiation callus number (piece) | Regeneration efficiency (%) |
| 321 | 321 | 136 | 42.4 |
In this example, the regeneration efficiency of callus regeneration system reaches 42.4%.
Example 3: phenylacetic acid shown in the associative list 3, inducing eucalyptus stem section produces the method that callus and differentiation are sprouted in this example, chooses by conventional method voluntarily a kind of alpine ash of breeding and 2 years living plant of Eucalyptus urophylla crossbreed, and all the other steps are identical with example 1.In this example, contain totally 396 of the somatic Albino Seedling stem explantses of differentiation, form 396 of callus through dedifferentiation, then callus differentiates indefinite bud again, totally 148.Specifically see Table 3:
Phenylacetic acid in table 3 example of the present invention
| Explant sum (piece) | Callus number (piece) | Differentiation callus number (piece) | Regeneration efficiency (%) |
| 396 | 396 | 148 | 37.4 |
In this example, the regeneration efficiency of this regenerative system reaches 37.4%.
By above-mentioned example as seen, the present invention utilizes the stem section of Albino Seedling, sprouts through the differentiation of callus regeneration approach, and regeneration rate is more than 30%.Correspondingly, callus of the present invention imports genes of interest through cultivating altogether, differentiation and bud formation again, the regeneration rate of transgenic calli also can reach more than 30%, thereby can realize eucalyptus, particularly Hybrid eucalyptus carry out genetic modification.
Claims (5)
1. an inducing eucalyptus produces callus and breaks up the method for sprouting, and may further comprise the steps:
1) gathers axillalry bud, carry out disinfection and rudiment;
2) root induction and Albino Seedling are induced cultivation, comprise following processing step:
A) with the 1st) step obtains the axillary bud sprouting bud and transfers to and contain 0.1-2mg/L 6-benzyl purine, induces the generation Multiple Buds on the MS medium of 0.01-1mg/L methyl α-naphthyl acetate;
B) the healthy and strong bud clump in the Multiple Buds is transferred on the 1/2MS medium that contains 0.1-1mg/L 3-indolebutyric acid and induces the seedling of taking root;
C) seedling of will taking root cuts away terminal bud, is seeded in to contain sucrose 30-100g/L, its NH
4NO
3Total content is 1650-6000mg/L, KH
2PO
4Total content is on the MS medium of 170-800mg/L, carries out in the dark Albino Seedling and induces cultivation;
3) induce cultivation take the stem section segment of Albino Seedling as explant and obtain callus, this calli induction media is for containing 0.2-2mg/L N-phenyl-N-1,2,3-thiadiazoles-5-urea, 0.01-0.1mg/L methyl α-naphthyl acetate, 50-200mg/L putrescine, the MS medium of 5-30mg/L spermidine;
4) with the 3rd) callus that produces of step transfers on the differential medium and cultivates, make the Calli Differentiation indefinite bud, described differential medium is to contain 0.2-2mg/L 6-benzyl purine, 0.05-0.5mg/L methyl α-naphthyl acetate, the 10-40mg/L putrescine, the MS medium of 0.5-3mg/L spermidine.
2. inducing eucalyptus as claimed in claim 1 produces callus and breaks up the method for sprouting, it is characterized in that: the described the 1st) step is the stem segment with axillary bud that gathers the livings eucalyptus Hybrid of 2-4, is seeded in that sprouting obtains aseptic bud on the MS medium after sterile-processed; The condition of culture of rudiment is: temperature 22-30 ℃, and unglazed photograph; The method of described sterilization is to adopt alcohol and mercuric chloride sterilization, uses aseptic water washing again.
3. inducing eucalyptus as claimed in claim 1 produces callus and breaks up the method for sprouting, and it is characterized in that: the 2nd) step specifically comprises:
A) cut the axillalry bud of certain-length, transfer to and be applied to mutually induce on the medium that produces Multiple Buds, cultivated 21-28 days, illumination 12-18 hour/day, intensity of illumination 1500-10000lx, temperature 22-30 ℃;
B) cut the bud that grows to certain-length, transfer to be applied to mutually induce take root and carried out root induction 15-30 days on the medium of seedling, illumination 12-18 hour/day, intensity of illumination 1500-10000lx, temperature 22-30 ℃;
C) seedling of will taking root cuts away terminal bud, keeps several axillalry buds of root and base portion, is seeded in to contain sucrose 30-100g/L, NH
4NO
34500mg/L, KH
2PO
4Carry out Albino Seedling on the MS medium of 500mg/L and induce cultivation, secretly cultivated temperature 22-30 ℃ 14-35 days.
4. inducing eucalyptus as claimed in claim 1 produces callus and breaks up the method for sprouting, and it is characterized in that: the described the 3rd) step specifically comprises:
A) take the stem section segment of Albino Seedling as explant, be seeded in and contain 0.05-0.3mg/L 2, the 4-dichlorphenoxyacetic acid carries out cell activation and cultivates on the MS medium of 0.1-0.5mg/L 6-benzyl purine, secretly cultivated temperature 22-30 ℃ 0-6 days;
B) explant after the activation is transferred to cultivate on the calli induction media and is produced callus, secretly cultivates temperature 22-30 ℃ 5-7 days;
C) transfer to the low light level according to cultivation 12-14 days, illumination 12-18 hour/day, intensity of illumination 20-200lx, 22-30 ℃.
5. inducing eucalyptus as claimed in claim 1 produces callus and breaks up the method for sprouting, and it is characterized in that: the described the 4th) step specifically comprises:
A) with the callus that obtains, transfer on the differential medium, transferred to subculture cultivation on the identical differential medium every 14-25 days, illumination 12-18 hour/day, intensity of illumination 500-3000lx, temperature 22-30 ℃;
B) subculture is cultivated and was produced indefinite bud in 14-100 days on differential medium.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201010249030 CN101897298B (en) | 2010-08-10 | 2010-08-10 | Method for inducing eucalyptus to generate calli and differentiating calli into buds |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201010249030 CN101897298B (en) | 2010-08-10 | 2010-08-10 | Method for inducing eucalyptus to generate calli and differentiating calli into buds |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101897298A CN101897298A (en) | 2010-12-01 |
| CN101897298B true CN101897298B (en) | 2013-04-10 |
Family
ID=43223497
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 201010249030 Expired - Fee Related CN101897298B (en) | 2010-08-10 | 2010-08-10 | Method for inducing eucalyptus to generate calli and differentiating calli into buds |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101897298B (en) |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102428873A (en) * | 2011-12-19 | 2012-05-02 | 普罗米绿色能源(深圳)有限公司 | Method for inducing adventitious buds of hybrid eucalyptus isolated organs through callus tissue differentiation |
| CN102524063A (en) * | 2011-12-19 | 2012-07-04 | 普罗米绿色能源(深圳)有限公司 | Method for effectively inducing regeneration of adventitious bud of eucalyptus stem segment in vitro |
| CN102577945A (en) * | 2012-01-31 | 2012-07-18 | 玉林市林业科学研究所 | Method for preventing gum trees from being vitrified in successive transfer culture |
| CN109380049B (en) * | 2018-10-26 | 2021-07-27 | 国家林业局桉树研究开发中心 | Eucalyptus composite cultivation method combining long-short cycle management |
| CN115039696B (en) * | 2022-05-09 | 2023-07-28 | 中国科学院华南植物园 | Method for regenerating seedlings by inducing callus of myrtle |
| CN116286964A (en) * | 2023-02-10 | 2023-06-23 | 中国林业科学研究院热带林业研究所 | Genetic transformation method of eucalyptus urophylla DH3213 |
| CN116472964B (en) * | 2023-05-12 | 2024-02-02 | 广东省林业科学研究院 | Eucalyptus differentiation medium and application thereof |
-
2010
- 2010-08-10 CN CN 201010249030 patent/CN101897298B/en not_active Expired - Fee Related
Non-Patent Citations (1)
| Title |
|---|
| 刘媛等.桉树再生体系的建立.《全国"植物生物技术及其产业化"研讨会论文摘要集》.2007, |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101897298A (en) | 2010-12-01 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101897298B (en) | Method for inducing eucalyptus to generate calli and differentiating calli into buds | |
| CN102301952B (en) | Method for breeding chamomile | |
| CN102870680B (en) | Efficient rapid propagation technique appropriate for detoxified rabbiteye blueberries | |
| CN102577969B (en) | Breeding method of tissue culture seedling of lonicera macranthoides Yulei No.1 | |
| CN102919125B (en) | Method for building efficient regeneration system of Yunnan rhododendron | |
| CN103155872B (en) | Eucalyptus urophylla*grandis embryoid induction seedling raising method | |
| CN101785428B (en) | Method for improving tissue culture reproductive speed of Alpinia zerumbet | |
| CN103026968A (en) | Factory production method of dendrobium officinale seedling | |
| CN103222425A (en) | Efficient and rapid propagation technology suitable for southern highbush blueberry | |
| CN102422815A (en) | Plant regeneration method by using giant tea rose stem segment as explant | |
| CN102228005A (en) | Pinellia ternate tissue culture one-step speciation method | |
| CN105850728B (en) | A kind of Jing Banxia seedling stems rapid propagation method | |
| CN104012417A (en) | High-efficiency and rapid micropropagation method for toxicodendron vernicifluum | |
| CN105379624B (en) | A kind of tissue culture and rapid propagation method of Eucalyptus pellita | |
| CN101491216B (en) | Evodia fruit tissue-culture quick propagation method | |
| CN113207690B (en) | Efficient one-step regeneration method taking paper mulberry root as explant | |
| CN102524065B (en) | High frequency regeneration method of Jatropha curcas | |
| CN104012416A (en) | Method for efficiently regenerating Sapium sebiferum leaf disc | |
| CN103340153A (en) | Tissue culture method taking masson pine in-vitro mature embryo as explant | |
| CN108243959A (en) | It is a kind of using yellow fine strain of millet wood stem section as the highly efficient regeneration method of explant | |
| CN101904302B (en) | Method for somatic cell embryogeny and plant regeneration of medicinal plant schisandga chinensis baill | |
| CN101836589B (en) | Method of rapid propagation of populus | |
| CN102524063A (en) | Method for effectively inducing regeneration of adventitious bud of eucalyptus stem segment in vitro | |
| CN110833028B (en) | Somatic embryogenesis and plant regeneration method for cinnamomum zhejiangense | |
| CN107258536A (en) | A kind of eucalyptus breeding tail eucalyptus camaldulensis DH191 7 tissue culture and rapid propagation method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C53 | Correction of patent of invention or patent application | ||
| CB03 | Change of inventor or designer information |
Inventor after: Li Hao Inventor after: Zhang Lanying Inventor after: Meng Lina Inventor after: Wang Lifang Inventor after: Jiang Shuzhen Inventor after: Chen Fang Inventor before: Li Hao Inventor before: Zhang Lanying Inventor before: Meng Lina Inventor before: Wang Lifang Inventor before: Jiang Shuzhen Inventor before: Chen Fang Inventor before: Li Ning |
|
| COR | Change of bibliographic data |
Free format text: CORRECT: INVENTOR; FROM: LI HAO ZHANG LANYING MENG LINA WANG LIFANG JIANG SHUZHEN CHEN FANG LI NING TO: LI HAO ZHANG LANYING MENG LINA WANG LIFANG JIANG SHUZHEN CHEN FANG |
|
| ASS | Succession or assignment of patent right |
Owner name: SINO-FOREST (GUANGZHOU) CO., LTD. Free format text: FORMER OWNER: PRIME GREEN ENERGY (SHENZHEN) CO., LTD. Effective date: 20120202 |
|
| C41 | Transfer of patent application or patent right or utility model | ||
| COR | Change of bibliographic data |
Free format text: CORRECT: ADDRESS; FROM: 518000 SHENZHEN, GUANGDONG PROVINCE TO: 5100000 GUANGZHOU, GUANGDONG PROVINCE |
|
| TA01 | Transfer of patent application right |
Effective date of registration: 20120202 Address after: Five million and one hundred thousand Citic Tower, No. 233 Tianhe North Road, Tianhe District, Guangdong, Guangzhou 2410A Applicant after: JIAHAN FORESTRY (GUANGZHOU) Co.,Ltd. Address before: 518000 Guangdong province Shenzhen city Nanshan District high tech park, Central West Road 25 2 3 floor West Applicant before: PROMETHEAN GREEN ENERGY (SHENZHEN) Co.,Ltd. |
|
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| ASS | Succession or assignment of patent right |
Owner name: SINO-FOREST (CHINA) INVESTMENTS CO., LTD. Free format text: FORMER OWNER: SINO-FOREST (GUANGZHOU) CO., LTD. Effective date: 20140331 |
|
| C41 | Transfer of patent application or patent right or utility model | ||
| COR | Change of bibliographic data |
Free format text: CORRECT: ADDRESS; FROM: 510000 GUANGZHOU, GUANGDONG PROVINCE TO: 510613 GUANGZHOU, GUANGDONG PROVINCE |
|
| TR01 | Transfer of patent right |
Effective date of registration: 20140331 Address after: 510613, room 2410-2411, Citic Tower, No. 233 Tianhe North Road, Tianhe District, Guangzhou, Guangdong Patentee after: SINO-FOREST (CHINA) INVESTMENTS LTD. Address before: 510000, Citic Tower, No. 233 Tianhe North Road, Tianhe District, Guangdong, Guangzhou 2410A Patentee before: JIAHAN FORESTRY (GUANGZHOU) Co.,Ltd. |
|
| CP01 | Change in the name or title of a patent holder |
Address after: 510613, room 2410-2411, Citic Tower, No. 233 Tianhe North Road, Tianhe District, Guangzhou, Guangdong Patentee after: Xinhan forestry investment (China) Co.,Ltd. Address before: 510613, room 2410-2411, Citic Tower, No. 233 Tianhe North Road, Tianhe District, Guangzhou, Guangdong Patentee before: SINO-FOREST (CHINA) INVESTMENTS LTD. |
|
| CP01 | Change in the name or title of a patent holder | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20130410 Termination date: 20210810 |