CN102668982B - Tissue culture method of mature wheat embryo - Google Patents
Tissue culture method of mature wheat embryo Download PDFInfo
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Abstract
The invention discloses a tissue culture method of a mature wheat embryo. The tissue culture method comprises the steps of seed disinfection, peeling of the embryo, inductive culture of callus tissues, differentiation culture and rooting culture, wherein culture media I and II are respectively adopted in the inductive culture of the callus tissues and the differentiation culture. The method disclosed by the invention can be used for obviously promoting the formation of the callus tissues and the occurrence of somatic embryos of the mature wheat embryo, obviously improving the callus induction rate and the differentiation rate, and further improving the regeneration rate of plants.
Description
Technical field
The present invention relates to field of plant tissue culture technique, specifically, relate to the method for tissue culture of one grained wheat mature embryo.
Background technology
One grained wheat (einkorn) is the dliploid kind (2n=2x=14, AA) of Triticum, is the important foundation species of wheat.One grained wheat has cold-resistant, characters of drought resistance, suitable to the mountain area cultivation, less demanding to soil, and find that it has the anti-source of Resistant Gene To Rust.Wheat is the hexaploid plant, has the characteristics such as genome is huge, repetitive sequence is many, Gene cloning is difficult.The dliploid one grained wheat is little than the Guard cell genome, is to insert mutagenesis to separate, to clone the good material of wheat cdna.Therefore, the one grained wheat Mature Embryo Tissue cultivation regenerating system of setting up efficient stable can not only provide reference for the dliploid Efficiency of Wheat Transformation, can also lay the foundation for Wheat Molecular Breeding research.
The foundation of Plant Tissue Breeding, particularly embryogenic cell line, the main foundation as the Genetic Transformation in Higher Plants operation is subject to extensive concern always.Up to the present, wheat class tissue is cultivated successful explant and is comprised rataria, young fringe, flower pesticide, mature embryo, spire, cotyledon middle level, seed, suspension cell, ovary, stem apex, Root apical meristem and protoplast etc.Study its Calli Differentiation situation, wherein, rataria is considered to optimal explant material, and callus induction rate and shoot regeneration frequency are all higher, obtains the report of Transgenic plant of wheat substantially take rataria as acceptor.And rataria is drawn materials and is subjected to the restriction in time, space and season, and suitable sampling time of explant is difficult to hold, and the physiological status of drawing materials, the uniformity of developmental stage can not finely ensure, have restricted to a certain extent effectively carrying out and application of genetic transformation.Mature embryo is drawn materials conveniently, is not subjected to the effects limit such as season, the stage of development of the plant, can guarantee the uniformity of physiological status between Different Individual, is to carry out the most convenient and practical acceptor of genetic transformation.At present, rarely have the report of setting up about wheat quasidiploid species tissue culturing systems, and the one grained wheat Mature Embryo Tissue is cultivated Establish of regeneration and still is in the starting stage.Therefore, demand setting up a kind of one grained wheat Mature Embryo Tissue cultivation regenerating system of efficient stable urgently, provide the basis for studying its genetic transformation.
Summary of the invention
The method for tissue culture that the purpose of this invention is to provide a kind of efficient one grained wheat mature embryo.
In order to realize the object of the invention, the method for tissue culture of one grained wheat mature embryo of the present invention, it comprises seed disinfection, peels off embryo, the step of induction of callus, differentiation cultivation and culture of rootage.
Wherein, the medium I that uses in described induction of callus step is: add in basal medium 2,4-dichlorphenoxyacetic acid (2,4-D) and/or the pH value of making after 6-kinetin (KT) be the medium of 5.8-6.0; The medium ii of using in described differentiation incubation step is: add the pH value made after one or more in α-naphthaleneacetic acid (NAA), 6-(the anti-cyclobutenyl of 4 hydroxy-3-methyls-2-) aminopurine (ZT), 6-benzyl aminopurine (6-BA), 6-kinetin (KT) or 2-ethoxy-trimethyl bursine (CC) and be the medium of 5.8-6.0 in basal medium.
Described basic culture solution formula is (with the water preparation):
Described basic culture solution formula is preferably (with the water preparation):
in preceding method, in the medium I that uses in described induction of callus step 2, the adding proportion of 4-D and KT is: 2, 4-D 0.5-1mg/L+KT 0-0.05mg/L, 2, 4-D1-2mg/L+KT 0-0.05mg/L, 2, 4-D 3-4mg/L+KT 0-0.05mg/L, 2, 4-D5-6mg/L+KT 0-0.05mg/L, 2, 4-D 0.5-1mg/L+KT 0.1-0.15mg/L, 2, 4-D1-2mg/L+KT 0.1-0.15mg/L, 2, 4-D 3-4mg/L+KT 0.1-0.15mg/L, 2, 4-D5-6mg/L+KT 0.1-0.15mg/L, 2, 4-D 0.5-1mg/L+KT 0.5-0.6mg/L, 2, 4-D 1-2mg/L+KT 0.5-0.6mg/L, 2, 4-D 3-4mg/L+KT 0.5-0.6mg/L or 2, 4-D 5-6mg/L+KT 0.5-0.6mg/L etc.
in preceding method, NAA in the medium ii of using in described differentiation incubation step, ZT, 6-BA, the adding proportion of KT and CC is: KT 0.5-1.0mg/L, KT 1.0-2.0mg/L, KT 2.0-3.0mg/L, NAA 0.5-1.0mg/L+KT 1.0-2.0mg/L, NAA0.5-1.0mg/L+ZT 1.0-2.0mg/L, NAA 0.5-1.0mg/L+6-BA 1.0-2.0mg/L, NAA 0.5-1.0mg/L+6-BA 1.0-2.0mg/L+KT 1.0-2.0mg/L, NAA0.5-1.0mg/L+6-BA 1.0-2.0mg/L+KT 1.0-2.0mg/L+ZT 1.0-2.0mg/L or NAA 0.5-1.0mg/L+6-BA 1.0-2.0mg/L+KT 1.0-2.0mg/L+ZT1.0-2.0mg/L+CC 1.0-2.0mg/L etc.
Preferably, described medium I is basal medium+2,4-D 1-2mg/L+KT0-0.05mg/L.Described medium ii is basic culture solution+NAA 0.5-1.0mg/L+ZT1.0-2.0mg/L.
In preceding method, the method of seed disinfection is: the one grained wheat mature seed is soaked 16-18 hour in water after, the alcohol that is placed in 75-80% was processed 0.5-1.5 minute, then was soaked in the mercuric chloride of 0.1-0.15% 13-15 minute, used at last aseptic water washing 3-5 time.
In preceding method, the method for induction of callus is: will peel off the mature embryo that obtains, scultellum is inoculated on medium I up, is placed in 24-25 ° of C and secretly cultivates, and obtains mature embryo callus.
In preceding method, the method that differentiation is cultivated is: mature embryo callus is transferred in medium ii, is placed in 25 ° of C, and illumination 16 hours/day, intensity of illumination is 2000-3000lux, is cultured to and emerges.
In preceding method, the method of culture of rootage is: the seedling that will grow after cultivating through differentiation is transferred in root media to be cultivated, height of seedling to be grown to reaches 6-9cm (as height of seedling 8cm, root system development is good) time, carry out hardening and (note moisturizing during hardening, temperature can not be greater than 30 ℃), after 3-5 days, plant moved into large Tanaka or carry out hot-house culture.
Wherein, the formula of described root media is (with the water preparation):
The invention has the advantages that: the method for tissue culture of one grained wheat mature embryo of the present invention, can significantly promote the formation of one grained wheat mature embryo callus and the generation of somatic embryo, and make healing rate and differentiation rate obtain remarkable improvement, further improve shoot regeneration frequency.
Description of drawings
Fig. 1 is the callus of induce situation of one grained wheat AS 260 mature embryos; Wherein, the mature embryo callus of induce situation of utilizing the inventive method that A and B gather respectively at 2011 and 2010, C is the mature embryo callus of induce situation of utilizing conventional method that gathers in 2011.
Fig. 2 is the callus of induce situation of one grained wheat AS 262 mature embryos; Wherein, the mature embryo callus of induce situation of utilizing the inventive method that A and B gather respectively at 2011 and 2010, C is the mature embryo callus of induce situation of utilizing conventional method that gathers in 2011.
Fig. 3 is the differentiation situation of one grained wheat AS 260 mature embryos; Wherein, the mature embryo differentiation situation of utilizing the inventive method that A and B gather respectively at 2011 and 2010, C is the mature embryo differentiation situation of utilizing conventional method that gathers in 2011.
Fig. 4 is the differentiation situation of one grained wheat AS 262 mature embryos; Wherein, the mature embryo differentiation situation of utilizing the inventive method that A and B gather respectively at 2011 and 2010, C is the mature embryo differentiation situation of utilizing conventional method that gathers in 2011.
Fig. 5 is the process chart that one grained wheat Mature Embryo Tissue of the present invention is cultivated renovation process.
Embodiment
Following examples are used for explanation the present invention, but are not used for limiting the scope of the invention.If do not specialize, the conventional means that in embodiment, technological means used is well known to those skilled in the art, the raw materials used commercial goods that is.
The tissue culture regenerating method of embodiment 1 one grained wheat mature embryo
1.1 the preparation of inducing culture (I) and differential medium (II)
Basic culture solution, its formula is (with the distilled water preparation):
The formula of callus inducing medium I is: basic culture solution+2,4-D 2mg/L, pH value 5.8.
The solution of above-mentioned preparation is placed in high-pressure sterilizing pot, 103.4kPa (1.05kg/cm
2), 121.3 ℃, sterilization in 15 minutes namely gets callus inducing medium I after cooling.
The formula of differential medium II is: basic culture solution+NAA 0.5mg/L+ZT 1.0mg/L, pH value 5.8.
The solution of above-mentioned preparation is placed in high-pressure sterilizing pot, 103.4kPa (1.05kg/cm
2), 121.3 ℃, sterilization in 15 minutes namely gets the differentiation medium ii after cooling.
1.2 seed disinfection is processed
From 5 kinds of one grained wheat genotype (AS 260, AS 262, AS 263, AS 265 and AS267, provided by Triticeae Research Institute, Sichuan Agricultural University) in, choose the one grained wheat mature seed of the full seed that gathered in 2011, after at room temperature soaking 16-18 hour with distilled water, alcohol disinfecting with 75% 1 minute, sterilized 15 minutes with 0.1% mercuric chloride again, use at last aseptic water washing 3-5 time.
1.3 peel off embryo
Strip embryo with the scalpel after sterilization.
1.4 induction of callus
Seed with after immersion treatment strips embryo with scalpel, and scultellum is inoculated on callus inducing medium I up, 40 mature embryos of every ware inoculation, and in 25 ° of C, 2 weeks of dark culturing, the formation of evoked callus.The callus growing state of one grained wheat AS 260 is (A and B gathered respectively at 2011 and 2010) as shown in Figure 1; The callus growing state of one grained wheat AS 262 is (A and B gathered respectively at 2011 and 2010) as shown in Figure 2.
1.5 differentiation is cultivated
Mature embryo callus with obtaining in 1.4 changes in differential medium II, light intensity 2000-3000lux, and light application time 16 hours/day, 25 ℃ of temperature cultivated for 3 weeks.The green seedling differentiation situation of one grained wheat AS 260 is (A and B gathered respectively at 2011 and 2010) as shown in Figure 3; The green seedling differentiation situation of one grained wheat AS 262 is (A and B gathered respectively at 2011 and 2010) as shown in Figure 4.
1.6 culture of rootage
The differentiation seedling that grows 2-3 sheet leaf is transferred in root media cultivated, light intensity 2000-3000lux, light application time 16 hours/day, 25 ℃ of temperature.Wait to grow to suitable size (height of seedling 8cm, root system development is good) after open test tube carry out hardening (during note moisturizing, temperature can not be greater than 30 ℃), be transplanted into flowerpot after 3-5d, then look ambient temperature and just determine to move into the greenhouse or continue growth in 24 ℃ of climatic cabinates.Measure respectively the regeneration rate (the mature embryo emergence rate=plant number of taking root/inoculation callus number) of the mature embryo of each material.Wherein, the regeneration rate of 5 kinds of one grained wheat genotype AS 260, AS 262, AS 263, AS 265 and AS 267 is respectively: AS 260 39.5%, AS 262 44.3%, AS 263 44.1%, AS 265 39.7%, AS 267 39.6%.
The formula of described root media is (with the distilled water preparation):
The solution of above-mentioned preparation is placed in high-pressure sterilizing pot, 103.4kPa (1.05kg/cm
2), 121.3 ℃, sterilization in 15 minutes namely gets root media after cooling.
Fig. 5 is the process chart that one grained wheat Mature Embryo Tissue of the present invention is cultivated renovation process.
The embodiment comparison of one grained wheat after utilizing the tissue culture regenerating method of mature embryo of the present invention of results respectively in 2 2009 years ~ 2011
1.1 according to method in embodiment 1 to 2009,2010 and 2011 respectively the one grained wheat of 5 kinds of genotype (AS 260, AS 262, AS 263, AS 265 and AS 267) of results carry out seed treatment, induce cultivations, differentiation cultivation and culture of rootage.
1.2 the situation comparative result that the seed maturity embryo tissue of different time results is cultivated is as shown in table 1:
The situation that the seed maturity embryo tissue of table 1 different time results is cultivated relatively
Can find out from table 1 and Fig. 1-Fig. 4, after the one grained wheat mature embryo of 5 kinds of different genotype of different time results is seeded on calli induction media I of the present invention, its embryo callus subculture healing rate and the equal no significant difference of emergence rate, but Light Difference is arranged between different genotype, wherein the highest with AS 262 and AS 263 healing rates, reached more than 70%, made emergence rate both also behave oneself best, reached more than 44%.Find simultaneously, the callus that utilizes the present invention to obtain, compact structure, color is vivid, and surface dry is laid a good foundation for further breaking up.
The method for tissue culture of embodiment 3 one grained wheat mature embryo of the present invention and the contrast of conventional tissue culture method
1.1 the tissue that utilizes the inventive method to carry out the Triticum tauschii mature embryo is cultivated regeneration
1.1.1 carry out seed treatment, induce cultivation, break up and cultivate and culture of rootage according to the one grained wheat of method in embodiment 1 to 5 kinds of genotype (AS 260, AS 262, AS 263, AS 265 and AS 267) of results in 2011.
Carry out one grained wheat Mature Embryo Tissue cultivation regeneration 1.2 utilize conventional wheat class Mature Embryo Tissue to cultivate renovation process
1.2.1 seed disinfection is processed
Choose equally 5 kinds of genotypic one grained wheat mature seeds of above-mentioned 2011 results, after at room temperature soaking 16-18 hour with distilled water, the alcohol disinfecting with 75% 1 minute, then sterilized 15 minutes with 0.1% mercuric chloride, use at last aseptic water washing 3-5 time.
1.2.2 peel off embryo
Strip embryo with the scalpel after sterilization.
1.2.3 induction of callus
Seed with after immersion treatment strips embryo with scalpel, and scultellum is inoculated on callus inducing medium up, 40 mature embryos of every ware inoculation, and in 25 ° of C, 2 weeks of dark culturing, the formation of evoked callus.Wherein, the formula of inducing culture is: basal medium+KT 1.0mg/L+2, and 4-D 3mg/L, pH 6.0.The callus growing state of one grained wheat AS 260 is as shown in Fig. 1 C; The callus growing state of one grained wheat AS 262 is as shown in Fig. 2 C.
1.2.4 differentiation is cultivated
With the mature embryo callus that obtains in 1.2.3, change in differential medium, light intensity 2000-3000lux, light application time 16 hours/day, 25 ℃ of temperature cultivated for 3 weeks.Consisting of of differential medium: basal medium+KT 1.0mg/L+6-BA 0.5mg/L+IAA (indolebutyric acid) 1.0mg/L, pH 6.0.The green seedling differentiation situation of one grained wheat AS 260 as shown in Figure 3 C; The green seedling differentiation situation of one grained wheat AS 262 is as shown in Fig. 4 C.
1.2.5 culture of rootage
The differentiation seedling that grows 2-3 sheet leaf is transferred in root media (formula is with the root media of implementing in 1) cultivated, light intensity 2000-3000lux, light application time 16 hours/day, 25 ℃ of temperature.Wait to grow to suitable size (height of seedling 8cm, root system development is good) after open test tube carry out hardening (during note moisturizing, temperature can not be greater than 30 ℃), be transplanted into flowerpot after 3-5d, then look ambient temperature and just determine to move into the greenhouse or continue growth in 24 ℃ of climatic cabinates.Measure respectively the regeneration rate (mature embryo regeneration ratio=regeneration plant number/inoculation callus number) of the mature embryo of each material.
1.2.6 the result that two kinds of methods are cultivated the one grained wheat Mature Embryo Tissue is as shown in table 2:
The result that two kinds of methods of table 2 are cultivated the one grained wheat Mature Embryo Tissue
As can be seen from Table 2, the one grained wheat mature embryo of 5 kinds of different genotype is seeded on different callus of induce and differential medium, and there are very big-difference in embryo callus subculture healing rate and final regeneration rate major part.Use method of the present invention, the mature embryo callus healing rate of one grained wheat on average improves 11.5% than conventional method, further impels the regeneration rate of using the inventive method on average to improve 11.8% than conventional method.Simultaneously, by Fig. 1 and Fig. 2 as seen, the callus that utilizes the inventive method to obtain, compact structure, color is vivid, surface dry, Differentiation is outstanding, for further differentiation is laid a good foundation.
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements, all belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (5)
1. the method for tissue culture of one grained wheat mature embryo comprises seed disinfection, peels off embryo, the step of induction of callus, differentiation cultivation and culture of rootage, it is characterized in that,
The medium I that uses in described induction of callus step is: add in basal medium 2,4-dichlorphenoxyacetic acid (2,4-D) and/or the pH value of making after 6-kinetin (KT) be the medium of 5.8-6.0; The medium ii of using in described differentiation incubation step is: basal medium+NAA 0.5-1.0mg/L+6-BA 1.0-2.0mg/L+KT 1.0-2.0mg/L+ZT 1.0-2.0mg/L, pH value 5.8-6.0;
The formula of described basal medium is:
NH
4NO
3 1600-1670 mg/L
KNO
3 1800-2000 mg/L
MgSO
4·7H
2O 370-380 mg/L
KH
2PO
4 160-180 mg/L
CaCl
2·2H
2O 420-440 mg/L
KI 0.8-0.9 mg/L
H
3BO
3 5-7 mg/L
MnSO
4·4H
2O 20-23 mg/L
ZnSO
4·7H
2O 8-9mg/L
Na
2MoO
4·2H
2O 0.2-0.25 mg/L
CuSO
4·5H
2O 0.02-0.025 mg/L
CoCl
2·6H
2O 0.02-0.025 mg/L
FeSO
4·7H
2O 27-28 mg/L
Na
2-EDTA·2H
2O 37-38 mg/L
Inositol 100-105 mg/L
Glycine 1-2 mg/L
Thiamine hydrochloride 0.05-0.1 mg/L
Puridoxine hydrochloride 0.4-0.5 mg/L
Nicotinic acid 0.4-0.5 mg/L
Proline 500-550 mg/L
Glutamine 500-550 mg/L
Asparagine 150-200 mg/L
Caseinhydrolysate 250-300 mg/L
Sucrose 55-60 g/L
Plant gel 2.4-2.5g/L
Prepare with water;
in the medium I that uses in described induction of callus step 2, the adding proportion of 4-D and KT is: 2, 4-D 0.5-1mg/L+KT 0-0.05mg/L, 2, 4-D 1-2mg/L+KT 0-0.05mg/L, 2, 4-D 3-4mg/L+KT 0-0.05mg/L, 2, 4-D 5-6mg/L+KT 0-0.05mg/L, 2, 4-D 0.5-1mg/L+KT 0.1-0.15mg/L, 2, 4-D 1-2mg/L+KT 0.1-0.15mg/L, 2, 4-D 3-4mg/L+KT 0.1-0.15mg/L, 2, 4-D 5-6mg/L+KT 0.1-0.15mg/L, 2, 4-D 0.5-1mg/L+KT 0.5-0.6mg/L, 2, 4-D 1-2mg/L+KT 0.5-0.6mg/L, 2, 4-D 3-4mg/L+KT 0.5-0.6mg/L or 2, 4-D 5-6mg/L+KT 0.5-0.6mg/L.
2. method according to claim 1, it is characterized in that, the method of seed disinfection is: the one grained wheat mature seed is soaked 16-18 hour in water after, the alcohol that is placed in 75-80% was processed 0.5-1.5 minute, then be soaked in the mercuric chloride of 0.1-0.15% 13-15 minute, use at last aseptic water washing 3-5 time.
3. method according to claim 2, is characterized in that, the method for induction of callus is: will peel off the mature embryo that obtains, scultellum is inoculated on medium I up, is placed in 24-25 ° of C and secretly cultivates, and obtains mature embryo callus.
4. method according to claim 3, is characterized in that, the method that differentiation is cultivated is: mature embryo callus is transferred in medium ii, is placed in 25 ° of C, and illumination 16 hours/day, intensity of illumination is 2000-3000 lux, is cultured to and emerges.
5. method according to claim 4, it is characterized in that, the method for culture of rootage is: the seedling that will grow after cultivating through differentiation be transferred in root media to be cultivated, when growing to height of seedling and reach 6-9cm, hardening 3-5 days, at last plant moved into large Tanaka or carry out hot-house culture; Wherein, the formula of described root media is:
NH
4NO
3 800-835 mg/L
KNO
3 900-1000 mg/L
MgSO
4·7H
2O 185-190 mg/L
KH
2PO
4 80-90 mg/L
CaCl
2·2H
2O 210-220 mg/L
KI 0.8-0.9 mg/L
H
3BO
3 5-6 mg/L
MnSO
4·4H
2O 20-23 mg/L
ZnSO
4·7H
2O 8-9 mg/L
Na
2MoO
4·2H
2O 0.2-0.25 mg/L
CuSO
4·5H
2O 0.02-0.025 mg/L
CoCl
2·6H
2O 0.02-0.025 mg/L
FeSO
4·7H
2O 27-28 mg/L
Na
2-EDTA·2H
2O 37-38 mg/L
Inositol 100-105 mg/L
Glycine 1-2 mg/L
Thiamine hydrochloride 0.05-0.1 mg/L
Puridoxine hydrochloride 0.4-0.5 mg/L
Nicotinic acid 0.4-0.5 mg/L
Sucrose 25-30 mg/L
Indolebutyric acid 0.5-1 mg/L
α-naphthaleneacetic acid 0.5-1 mg/L
Paclobutrazol 0.5-1 mg/L
Plant gel 2.4-2.5 g/L
Prepare with water.
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| CN103210843B (en) * | 2013-04-09 | 2014-09-24 | 南京农业大学 | High-frequency roegneria kamoji immature embryo callus induction and regeneration cultivation method |
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