CN102220372B - Method for obtaining monthly rose samantha transgenic plant - Google Patents
Method for obtaining monthly rose samantha transgenic plant Download PDFInfo
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- CN102220372B CN102220372B CN 201110099816 CN201110099816A CN102220372B CN 102220372 B CN102220372 B CN 102220372B CN 201110099816 CN201110099816 CN 201110099816 CN 201110099816 A CN201110099816 A CN 201110099816A CN 102220372 B CN102220372 B CN 102220372B
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Abstract
The invention discloses a method for obtaining monthly rose samantha transgenic plant. The method comprises the following steps of: adopting somatic embryo obtained through the induction of immature leaves of monthly rose samantha as a receptor, and carrying out the processes of somatic embryo induction, subcultivation multiplication cultivation for transformation receptor, genetic transformation, cocultivation, selective enrichment cultivation, selective germination cultivation, selective seedling cultivation through the agrobacterium mediated method, thereby finally obtaining the monthly rose samantha transgenic plant. The method achieves simplicity and a shorter cultivation period, successfully realizes the cultivation of the monthly rose samantha transgenic plant and fills in the gap of cultivation of the monthly rose samantha transgenic plant.
Description
Technical field
The invention belongs to the plant transgenic technology field, being specifically related to Chinese rose ' Sa Mansha ' somatic embryo is acceptor, through the agriculture bacillus mediated method of carrying out the Chinese rose genetic transformation and obtaining Chinese rose ' Sa Mansha ' transfer-gen plant.
Background technology
Chinese rose (Rosa hybrida) belongs to the Rosaceae (Rosaceae) rose (Rosa) perennial evergreen xylophyta.The Chinese rose cultivation history is long; Extensively planted by countries in the world, be very important ornamental flower and have important commercial and be worth, but since the bottle of Flos Rosae Chinensis to insert the life-span shorter; Be prone to suffer from Powdery Mildew, black spot, these have all influenced its ornamental value and economic worth.On these character improvements, traditional breeding way exists significant limitation.Plant gene engineering technology can utilize foreign gene that its proterties of controlling is carried out orderly improvement on the metastable basis of other proterties that keeps kind, thereby for cultivating the Chinese rose new variety new approach is provided.
The prerequisite of plant being carried out genetic transformation is to have a reliable and effective regeneration system rapidly.At present the regeneration system of Chinese rose is also only perfect, and most regeneration rate is not high and obviously receive genotypic restriction, so the successful report of relevant Chinese rose genetic transformation is very limited.First piece of report about the Chinese rose genetic transformation is Firoozabady et al. (1991) with the embryo callus of R.hybrida cv.Royalty is acceptor, has obtained transformed plant through agrobacterium mediation method.Subsequently; The adventive root that Van der Salm et al. (1997) has utilized internode tissue and the Agrobacterium of R.hybridacv.Moneyway on the substratum of root induction, to cultivate altogether to obtain transforming; Differentiation at inducing adventitious root produces embryoid, thereby obtains the regenerated transformed plant.Subsequently, utilize the agriculture bacillus mediated research of carrying out the Chinese rose genetic transformation to become focus, and many pieces of successfully report (Li et al., 2002b are arranged; 2003).Marchant etal. (1998a) adopts particle bombardment; With the embryo callus is the bombardment object; Successfully the chitinase gene with paddy rice has been transferred among the R.hybrida cv.Glad Tidings, thereby makes the black spot occurrence frequency of this kind reduce 13%-43%.The Chinese rose genetic transformation also has the successful report that utilizes the PEG mediated method; Wherein the protoplastis factors such as condition and foreign DNA of separating source, the cell suspension culture of used mixture, plant gene type and experiment material all produce significant effects (Schum and Hofmann, 2001) to the result of its conversion.Gao etal. (2004) when modern rose cultivars ' Sa Mansha ' is carried out agriculture bacillus mediated genetic transformation, expresses as the moment that transformation receptor does not detect gus gene with blade; Though obtained containing the callus of reporter gene and goal gene with callus during as transformation receptor, finally do not obtained plants transformed.Li Jingrui (2006) has obtained resistant buds with blade as transformation receptor, but still has not obtained transformed plant when modern rose cultivars ' Sa Mansha ' is carried out agriculture bacillus mediated conversion.
The patented claim of Hua Zhong Agriculture University " is the method for the Chinese rose regeneration plant of explant with the blade " (201010256892.3); Proposition is an explant with Chinese rose prematurity blade; Through the somatocyte embryogenesis path; Set up the method for Chinese rose regeneration system, but it does not have the something lostization step of converting, can't realize breed improvement.
At present, both at home and abroad also not about obtaining the successful report of Chinese rose ' Sa Mansha ' transfer-gen plant.
Summary of the invention
The objective of the invention is in order to solve the problems of the technologies described above; Providing a kind of is explant with Chinese rose ' Sa Mansha ' prematurity blade; With the somatic embryo is transformation receptor, through agriculture bacillus mediated method of carrying out genetic transformation acquisition transfer-gen plant, has the advantage that method is simple, culture cycle is short.
The present invention obtains the method for Chinese rose ' Sa Mansha ' transfer-gen plant, and inducing the somatic embryo that obtains with Chinese rose ' Sa Mansha ' prematurity blade is acceptor, through agrobacterium mediation method, handles by following step:
(1) the body blastula is induced: with Chinese rose prematurity blade is explant, and described explant is directly induced somatic embryo in the somatic induction substratum;
(2) shoot proliferation of transformation receptor is cultivated: the somatic embryo that induces is inoculated in the somatic embryo shoot proliferation substratum, for genetic transformation provides the transformation receptor material;
(3) genetic transformation: will change over to as the transformation receptor material in the Agrobacterium bacterium liquid for preparing through the somatic embryo that shoot proliferation is cultivated and infect, said time of infection is 40-45min;
(4) somatic embryo after will infecting then carries out common cultivation, selects multiplication culture, selects to sprout and cultivate, be chosen to the transfer-gen plant that Chinese rose ' Sa Mansha ' cultivated and finally obtained to seedling.
Somatic induction medium component in the said step (1) comprises: MS minimum medium, 2, and 4-D 3.0-4.0mg/L, glucose 30.0g/L, GEL 2.5mg/L, adding distil water is to 1L, pH 6.0.
Somatic embryo shoot proliferation medium component comprises in the said step (2): MS minimum medium, 2; 4-D 1.0mg/L, 6-BA 0.01-0.05mg/L, glucose 45.0-60.0g/L, GEL 2.5mg/L; Adding distil water is to 1L; PH 6.0, and per 4 weeks are upgraded subcultures, and are used in the shoot proliferation substratum of renewal and cultivated the acceptor of the somatic embryo in 2 weeks as genetic transformation
Immerged time is 40min in the said step (3).
In the said step (4); For the somatic embryo after will infecting changes in the common culture medium; Under 24 ± 2 ℃ of dark conditions, carry out common cultivation 3d; Change somatic embryo then over to and select to select 8 weeks of multiplication culture in the proliferated culture medium, per two weeks are upgraded a subculture, and screening obtains the resistance somatic embryo; Said selection is sprouted to cultivate to the resistance somatic embryo that screening is obtained is inoculated into somatocyte and is selected to cultivate for 8 weeks in the germination medium, and per two weeks are upgraded a subculture, and screening obtains resistant buds; The said seedling that is chosen to is cultivated to the resistant buds that screening is obtained is inoculated into and is chosen in the seedling substratum, upgrades a subculture around every, finally obtains the transfer-gen plant of Chinese rose ' Sa Mansha '.
In the said step (4), said culture medium composition altogether comprises: the MS minimum medium, and 2,4-D 1.0mg/L, 6-BA 0.01mg/L, AS100 μ mol/L, glucose 45.0g/L, GEL 2.5mg/L, adding distil water are to 1L, and pH 6.0; Said somatic embryo selects the proliferated culture medium composition to comprise: the MS minimum medium, and 2,4-D 1.0mg/L, 6-BA 0.01mg/L, Km80-100mg/L, Cef300mg/L, glucose 45.0g/L, GEL 2.5mg/L, adding distil water are to 1L, and pH 6.0; Said somatocyte selects the germination medium composition to comprise the MS minimum medium, BA1.0mg/L, NAA 0.1mg/L, GA
30.5mg/L, Km50-75mg/L, Cef300mg/L, glucose 30.0g/L, GEL 2.5mg/L, adding distil water are to 1L, and pH 6.0; The said seedling substratum composition that is chosen to comprises: MS minimum medium, BA1.0mg/L, NAA 0.1mg/L, GA
30.5mg/L, Km0-30mg/L, Cef300mg/L, glucose 30.0g/L, GEL2.5mg/L, adding distil water are to 1L, and pH 6.0.
Described Km is kantlex (Kanamycin): the mother liquor that is mixed with 100mg/L with sterile distilled water; Cef is cephamycin (Cefotaxime): be mixed with the mother liquor of 200mg/L with sterile distilled water, AS is a Syringylethanone: take by weighing a certain amount of AS, the alcohol dissolving with 95%; Use the sterile distilled water constant volume at last; Be mixed with the mother liquor of 10 μ mol/ml, more than three kinds of biochemical reagents all adopt the sterilization of 0.45 μ m membrane filtration, in-20 ℃ of preservations;
Described MS is a minimum medium, 2, and 4-D is a 2,4 dichlorophenoxyacetic acid, BA is a 6-benzyladenine, NAA naphthylacetic acid, GA
3Be Plant hormones regulators,gibberellins, Glucose is a glucose, and GEL is a plant gel.
For the ease of to understanding of the present invention, the applicant has done as giving a definition used plant hormone in substratum used in above-mentioned each step, the substratum: substratum comprises: somatic embryo inducement substratum, somatic embryo shoot proliferation substratum, culture medium, somatic embryo are selected proliferated culture medium, select germination medium, are chosen to the seedling substratum altogether.Minimum medium used herein is originated as follows: and the MS minimum medium (Murashige T.and F.Skoog.Physiol.Plant, 1962,15:473-497).
Owing to be as the transformation receptor material with the somatic embryo cultivated through shoot proliferation; Its genetic transformation control condition and existing embryo callus are that acceptor material carries out genetic transformation and also is not quite similar; Research shows ' Sa Mansha ' kind to Chinese rose; Infection condition with can obtain the resistance somatic embryo extremely important relation is arranged, must strict control time of infection at 40-45min, preferred 40min. time of infection is too short; Agrobacterium is not adsorbed onto the wound or the tissue surface of acceptor material as yet, thereby is unfavorable for that growth of Agrobacterium reduces genetic transformation efficiency; Time of infection is long, makes brownization of acceptor material even death thereby Agrobacterium produces toxic action to acceptor material.The bacterial concentration of Agrobacterium is with reference to existing bacterial concentration, and preferred bacterial concentration is OD value 0.6-0.8, and suitable and time of infection and bacterial concentration can effectively improve changing effect; Further; The applicant is through research for many years, experiment is found repeatedly, the selection multiplication culture in the step (4), selects to sprout and cultivates, is chosen in the seedling culturing process that the control of kantlex content also has material impact to improving changing effect in the corresponding substratum, therefore; Kantlex (Km) content is 80-100mg/L in the somatic embryo selection proliferated culture medium; Preferred 100mg/L, kantlex (Km) content is 50-75mg/L in the selection germination medium, preferred 50mg/L; Being chosen to seedling substratum kantlex (Km) content is 0-30mg/L, preferred 30mg/L.
The present invention utilizes the somatic embryo of cultivating through shoot proliferation as the transformation receptor material; Can directly select multiplication culture after infecting; Reduced process step, more existing is that acceptor material carries out the culture cycle that genetic transformation obtains the transfer-gen plant method and compares with the embryo callus, can reduce the 4-8 time-of-week; Thereby shortened culture cycle, and ' Sa Mansha ' transfer-gen plant that successfully obtained Chinese rose.
Beneficial effect:
1, realizes successfully that through the inventive method with Chinese rose ' Sa Mansha ' somatic embryo be the transformation receptor material; Through the agriculture bacillus mediated and further transfer-gen plant that has obtained to contain gus reporter gene of cultivating of later stage, for the conversion of goal gene is laid a good foundation.
2, technology is simple, culture cycle is shorter.
3, the explant source that the present invention adopted does not receive season limit, can carry out tissue and the cell cultures of Chinese rose ' Sa Mansha ' the anniversary.
4, the Chinese rose under the isolated culture ' Sa Mansha ' somatic embryo also can constantly be bred through secondary embryo, has good cultivation effect.
Description of drawings
Fig. 1: Chinese rose among the present invention ' Sa Mansha ' somatic embryo is the genetic transformation technological line figure of acceptor.
Fig. 2: the GUS of the rotaring gene plant blade of Chinese rose among the present invention ' Sa Mansha ' is active to be detected, the negative contrast of a, and b, c are the blade of transfer-gen plant,
Fig. 3: d is the blade enlarged view of transfer-gen plant.
Embodiment
Test materials is selected, substratum designs and inoculation culture
1, test materials source and processing thereof:
Chinese rose ' Sa Mansha ' prematurity blade is induced the somatic embryo that obtains, and somatic embryo is inoculated in and carries out the proliferation and subculture cultivation in the somatic embryo proliferated culture medium, is acceptor material to change the somatic embryos of cultivating for 2 weeks in the fresh proliferated culture medium over to;
Bacterial strain and plasmid: used agrobacterium tumefaciens is the C58 bacterial strain, contains the pCAMBIA2301 plasmid, and this plasmid carries gus reporter gene behind the 35S composition type expression promoter, and merges Km resistance screening gene.
2, substratum design:
Table 1 has been listed the composition and the consumption thereof of various substratum of the present invention.
The isolated culture base design of table 1 Chinese rose
Annotate: the preparation of MS minimum medium is referring to Murashige T.and F.Skoog.Physiol.Plant, 1962,15:473-497.
Plant hormones regulators,gibberellins (GA in the table 1
3), kantlex (Kanamycin, Km), (Cefotaxime Cef), Syringylethanone (AS) all adopts 0.45 μ m membrane filtration sterilization, adds behind the substratum high-temperature sterilization cephamycin.
The code name of various compositions is following in the substratum: and 2,4 dichlorophenoxyacetic acid (2,4-D), 6-benzyladenine (6-BA), naphthylacetic acid (NAA), Plant hormones regulators,gibberellins (GA
3), kantlex (Kanamycin, Km), cephamycin (Cefotaxime, Cef), Syringylethanone (AS), plant gel (GEL) all can be from commercial purchase.
3, culture condition
24 ± 2 ℃ of culturing room's culture temperature, intensity of illumination 1000-1500lx, periodicity of illumination are 14h; 24 ± 2 ℃ of dark culturing temperature.
4, the preparation of GUS dye liquor
The 50mmol/L sodium phosphate buffer contains in (pH 7.0): 0.1mol/L K
3[Fe (CN)
6], 0.1mol/L K
4[Fe (CN)
6], 10mmol/L Na
2EDTA, 0.001% (v/v) TritonX-100,20% methyl alcohol, 0.5mg/L X-Gluc.(reference: plant genetic engineering, Wang Guanlin, Fang Hongjun chief editor)
5, genetic transformation process:
1. the preparation of acceptor material: with Chinese rose prematurity blade is explant; Described explant is directly induced somatic embryo in inducing culture; Somatic embryo is inoculated in the somatic embryo shoot proliferation substratum; Per 4 weeks are upgraded subcultures, and are used in the shoot proliferation substratum of renewal and have cultivated the acceptor of the somatic embryo in 2 weeks as genetic transformation, for genetic transformation provides acceptor material;
2. the preparation of bacterial strain: from-70 ℃ of refrigerators, take out the bacterial classification of being preserved, rule containing on the LB solid medium of 100mg/L Km, then flat board is inverted in that dark culturing produces until single bacterium colony in 28 ℃ of incubators with transfering loop.Picking list colony inoculation is in containing 100mg/L KmLB liquid nutrient medium, and the 180r/min shaking culture is spent the night on 28 ℃ of constant temperature shaking tables.OD value with logarithmic phase changes over to from triangular flask in the aseptic 50ml centrifuge tube for 0.6-0.8 bacterium liquid then; The centrifugal 8min of 5000r/min; Remove supernatant; The Agrobacterium thalline of collecting is placed the MS liquid nutrient medium that contains 100 μ M AS, behind the resuspended cultivation of 180r/min on 28 ℃ of constant temperature shaking tables 2h, be used for infecting of transformation receptor;
3. infect: step is cultivated the robust growth in two weeks in 1. in proliferated culture medium somatic embryo changes the bacterium liquid for preparing over to and infects 40-45min;
4. cultivate altogether: the somatic embryo that will infect is inoculated in the common culture medium after on filter paper, removing bacterium liquid, places 24 ± 2 ℃, and dark condition is cultivated 3d down;
5. select to cultivate: the somatic embryo after will cultivating altogether is inoculated into somatic embryo and selects to cultivate for 8 weeks in the proliferated culture medium, and per two weeks are upgraded a subculture; The resistance somatic embryo that screening obtains is inoculated into to be selected to cultivate for 8 weeks in the germination medium, and per two weeks are upgraded a subculture; The resistant buds inoculation that screening obtains is chosen in the seedling substratum, resistant buds regeneration plant, every subculture that upgrades all around.
6. the blade with rotaring gene plant blade and unconverted plant is a material, carries out the active detection of GUS, the negative contrast of the blade of unconverted plant; With the transformed plant blade is material, adopts the CTAB method to extract total DNA, carries out PCR and detects.
6, test-results
The somatic embryo that transforms in the test adds up to 720, detects and the PCR detection through GUS is active, obtains 17 transfer-gen plants altogether, and transformation efficiency is 2.36%.
Claims (2)
1. method that obtains Chinese rose ' Sa Mansha ' transfer-gen plant, it is characterized in that: inducing the somatic embryo that obtains with Chinese rose ' Sa Mansha ' prematurity blade is acceptor, through agrobacterium mediation method, handles by following step:
(1) the body blastula is induced: with Chinese rose prematurity blade is explant, and described explant is directly induced somatic embryo in the somatic induction substratum;
(2) shoot proliferation of transformation receptor is cultivated: the somatic embryo that induces is inoculated in the somatic embryo shoot proliferation substratum, for genetic transformation provides the transformation receptor material;
(3) genetic transformation: will change over to as the transformation receptor material through the somatic embryo that shoot proliferation is cultivated in the bacterium liquid of Agrobacterium preparation and infect, said time of infection is 40-45min;
(4) somatic embryo after will infecting then carries out common cultivation; Select multiplication culture, select to sprout and cultivate, be chosen to the transfer-gen plant that Chinese rose ' Sa Mansha ' cultivated and finally obtained to seedling; Be specially: the somatic embryo after will infecting earlier changes in the common culture medium, under 24 ± 2 ° of C dark conditions, carries out common cultivation 3d, changes somatic embryo then over to and selects to select 8 weeks of multiplication culture in the proliferated culture medium; Per two weeks are upgraded a subculture, and screening obtains the resistance somatic embryo; Said selection is sprouted to cultivate to the resistance somatic embryo that screening is obtained is inoculated into somatocyte and is selected to cultivate for 8 weeks in the germination medium, and per two weeks are upgraded a subculture, and screening obtains resistant buds; The resistant buds that then screening is obtained is inoculated into and is chosen in the seedling substratum, whenever upgrades a subculture all around, finally obtains the transfer-gen plant of Chinese rose ' Sa Mansha ';
Somatic induction medium component in the said step (1) comprises: MS minimum medium, 2, and 4-D 3.0-4.0mg/L, glucose 30.0g/L, GEL 2.5 mg/L, adding distil water is to 1L, pH 6.0;
Somatic embryo shoot proliferation medium component comprises in the said step (2): MS minimum medium, 2; 4-D 1.0mg/L, 6-BA 0.01-0.05mg/L, glucose 45.0-60.0g/L, GEL 2.5 mg/L; Adding distil water is to 1L; PH 6.0, and per 4 weeks are upgraded subcultures, and are used in the shoot proliferation substratum of renewal and cultivated the acceptor of the somatic embryo in 2 weeks as genetic transformation;
In the said step (4), said culture medium composition altogether comprises: the MS minimum medium, and 2,4-D 1.0mg/L, 6-BA 0.01mg/L, AS100 μ mol/L, glucose 45.0g/L, GEL 2.5 mg/L, adding distil water are to 1L, and pH 6.0; Said somatic embryo selects the proliferated culture medium composition to comprise: the MS minimum medium, and 2,4-D 1.0mg/L, 6-BA 0.01mg/L, Km80-100mg/L, Cef300mg/L, glucose 45.0g/L, GEL 2.5 mg/L, adding distil water are to 1L, and pH 6.0; Said somatocyte selects the germination medium composition to comprise the MS minimum medium, BA1.0mg/L, NAA 0.1 mg/L, GA
30.5mg/L, Km50-75mg/L, Cef300mg/L, glucose 30.0g/L, GEL 2.5 mg/L, adding distil water are to 1L, and pH 6.0; The said seedling substratum composition that is chosen to comprises: MS minimum medium, BA1.0mg/L, NAA 0.1 mg/L, GA
30.5mg/L, Km0-30mg/L, Cef300mg/L, glucose 30.0g/L, GEL 2.5 mg/L, adding distil water are to 1L, and pH 6.0.
2. the method for acquisition Chinese rose ' Sa Mansha ' transfer-gen plant as claimed in claim 1, it is characterized in that: immerged time is 40min in the said step (3).
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| CN106868042B (en) * | 2017-04-20 | 2020-04-03 | 南京农业大学 | Vacuum infiltration transgenic method for Chinese rose adventitious bud |
| CN113025645B (en) * | 2021-03-11 | 2024-04-26 | 武汉隽秀园艺科技有限公司 | Method for obtaining gypsophila transgenic plant by taking callus as receptor |
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| CN1900291A (en) * | 2006-07-24 | 2007-01-24 | 华中农业大学 | Method for cultivating transgenic sycamore plant mediated by agrobacterium |
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Non-Patent Citations (3)
| Title |
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| 高莉萍，包满珠.月季的植株再生及遗传转化研究进展.《植物学通报》.2005,第22卷(第2期),231-237. * |
| 高莉萍.优化农杆菌介导的月季遗传转化系统的研究.《北京林业大学学报》.2005,第27卷(第4期),60-64. * |
| 高莉萍.月季品种‘萨蔓莎’植株再生体系的建立和根癌农杆菌介导的遗传转化研究.《中国博士学位论文全文数据库（农业科技辑）》.2006,(第3期),1-82. * |
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