CN102550414A - Transgenic cultivation method of rice dedicated for starch medium - Google Patents
Transgenic cultivation method of rice dedicated for starch medium Download PDFInfo
- Publication number
- CN102550414A CN102550414A CN2012100051019A CN201210005101A CN102550414A CN 102550414 A CN102550414 A CN 102550414A CN 2012100051019 A CN2012100051019 A CN 2012100051019A CN 201210005101 A CN201210005101 A CN 201210005101A CN 102550414 A CN102550414 A CN 102550414A
- Authority
- CN
- China
- Prior art keywords
- starch
- medium
- callus
- rice
- screening
- 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.)
- Pending
Links
Images
Abstract
The invention relates to a transgenic cultivation method of rice dedicated for a starch medium. The method comprises the following steps: a mature embro of a rice species with a low content of alpha amylose is taken as an explant and is induced to form calluses; the calluses are subcultured to enter a proliferation period and are preculutured under a dark condition; the calluses are co-cultivated with agrobacterium tumefaciens for transformation and then transferred into a screening culture medium for being screened for three tims; resistant calluses are taken to be induced and differentiated into seedlings, and histochemical stain and molecular identification are performed to resistant plants to identify the differentiated plants containing target genes; the starch gelanitization property of selected transgenic plants is identified, and the transgenic plants which have the starch gelatinized and can form semi-solid gel after being cooled are selected and reserved; and expression stability of the starch gelatinization property of the plants is further evaluated, seed propagation is performed to the excellent plants with a stable starch gelatinization property, and finally dedicated rice for the starch medium is cultivated. The dedicated rice for the starch medium can be taken as a novel biological medium and be widely applied to scientific researches, food, medicine and chemical engineering.
Description
Technical field
The present invention relates to a kind of transgenic culturing method of starch matrix rice special.
Background technology
Whole nation tissue culture room surpasses 1000, and tissue culture technique is widely used in nearly 400 kinds of economic plants such as crops, fruit tree, vegetables, forest, ornamental plants and traditional Chinese medicine, has obtained the huge social economic benefit at aspects such as fast numerous and detoxifications.Too high because of cost, the commercial exploitation of tissue culture receives very big restriction, can't meet the demand of market to high-quality tissue cultivating seedling, low price cost.Therefore, reducing cost is the more commercial most important condition of success of tissue culture technology.
Aspect the culture medium cost that reduces tissue culture, the methods that adopt the increase reproduction coefficient more, but the increase of reproduction coefficient causes the reduction of tissue cultivating seedling quality, and not only the ratio of glass seedling significantly improves, and has a strong impact on its follow-up growth.Agar is most widely used a kind of coagulating agent in the present various biological medium, and not only consumption is huge and be the most expensive a kind of in all medicines, accounts for more than 70% of cost of drugs.Only the market price of group training level agar just up to 160 yuan/more than the kg, every liter of the cost of using always is just up to more than 8 yuan.
Starch is the nutrient of storing in the plant corpus, is the important component part of food, and is starch-containing 62% ~ 86% in the paddy rice, but that the starch of common rice kind solidifies resistance is very low, and colloform texture is unstable, is difficult to directly or the modification utilization.Amylose heats gelatinization in water after, unstable and meeting is worn out rapidly and is progressively formed gelinite, but this gelinite is harder, needs high temperature just oppositely to transform; Amylopectin is stable in the aqueous solution, gels-soft, and its intensity of gel is reversible with variations in temperature.Starch branch enzyme gene sbeIIb is the synthetic key enzyme of regulation and control starch, can optimize and promote the synthetic of amylose, realizes the remarkable improvement of amylose and gel characteristic thereof.
Summary of the invention
Under above-mentioned background, the purpose of this invention is to provide a kind of transgenic culturing method of starch matrix rice special just, select for use the low amylose content long-grained nonglutinous rice to be the basis; Promote the synthetic of amylose through starch branch enzyme gene optimization; Obtain the starch gel performance and take into account the paddy rice of straight chain and side chain double grading, high resistance and the stable novel starch matrix of colloform texture of coagulating of initiative substitutes agar; The development new bio is cultivated bio-matrix
Be widely used in scientific research, medical science and chemical industry.
The transgenic culturing method of starch matrix rice special of the present invention may further comprise the steps:
1) cut-off chain content of starch is an explant between the mature embryo of 10% ~ 15% rice varieties, is seeded to
Inducing culture is induced the formation callus under 25 ± 1 ℃, light intensity 3000 lux optical condition;
2) will induce the callus that formed for 2 weeks, and be transferred to subculture medium, successive transfer culture to callus gets into breeds the phase at double;
3) callus that will breed at double carries out the preparatory cultivation 2 days of acceptor callus under 25 ± 1 ℃, the dark condition of lucifuge;
4) will pass through pre-incubated callus, in agrobacterium suspension, soak 10 minutes, discard bacterium liquid afterwards, blot, be inoculated on the common medium, under 25 ± 1 ℃, the dark condition of lucifuge, cultivate altogether 2 days with aseptic filter paper;
5) with above-mentioned cultured calli altogether, after cleaning, drying, transfer to screening culture medium; Under 25 ± 1 ℃, the dark condition of lucifuge, cultivated for 6 weeks, collect the resistant calli that newly grows, be transferred on the new screening culture medium; Carry out continuous 2 again and take turns the subculture screening, whenever continue 1 time at a distance from 3 turnovers;
6) get 3 resistant callis of taking turns after the screening; Be transferred to differential medium; Under 25 ± 1 ℃, light intensity 3000 lux optical condition, induce seedling differentiation; The antagonism plant carries out histochemical stain and Molecular Detection, select and remain contain the genes of interest insetion sequence transfer-gen plant, said genes of interest insetion sequence is meant the insetion sequence that comprises genes of interest starch branch enzyme gene sbeIIb, marker gene glucuronidase gene GUS and the anti-hygromycin gene HPT of screening-gene.Genes of interest starch branch enzyme gene sbeIIb sheet segment length 643bp;
7) be taken into the transfer-gen plant of choosing, gather in the crops seed behind the field planting, identify the starch gel performance, can form the transfer-gen plant of semisolid gel after the starch gelatinization of the selecting and remain cooling;
8) continue the transgenic line that the plantation step 7) is screened, the starch gel resistance expression stability of evaluation, the elite plant strain stable to the starch gel resistance carries out seminal propagation, is the starch matrix rice special of cultivating.
Among the present invention, said inducing culture adds 2 for the N6 minimal medium, 4-D1.5mg, sucrose 30g and agar powder 6.8g, pH sterilization preceding 5.8.
Among the present invention, said subculture medium adds 2 for the N6 minimal medium, 4-D 1mg sucrose 30g and agar powder 6.8g, pH sterilization preceding 5.8.
Among the present invention, said medium altogether adds 2 for the N6 minimal medium, 4-D 0.5mg, acetosyringone 1mg, sucrose 30g and agar powder 6.8g, pH sterilization preceding 5.8.
Among the present invention, said screening culture medium adds 2 for the N6 minimal medium, 4-D 0.5mg, hygromycin 30mg, carboxylic Bian 300 mg, sucrose 30g and agar powder 6.8g, pH sterilization preceding 5.8.
Among the present invention, said differential medium adds 6-BA1mg, NAA0.5mg, KT0.5mg, carboxylic Bian 200mg, sucrose 30g and agar powder 6.8g for the N6 minimal medium, pH sterilization preceding 5.8.
Above-mentioned N6 minimal medium is KNO
32830mg, (NH
4)
2SO
4463mg, CaCl
22H
2O 166mg, MgSO
47H
2O 185mg, KH
2P0
4400mg, MnSO
44H
2O 4.4mg, ZnSO
47H
2O 1.5mg, FeSO
47H
2O 27.8mg, Na
2-EDTA2H2O 37.3mg, H
3BO
31.6mg, inositol 100mg, nicotinic acid 0.5mg, vitamin B6 0.5mg, vitamin B1 0.5mg and glycine 2mg.
Among the present invention; The bacterial strain of said Agrobacterium is EHA105; Include plasmid pCAM1305, the insetion sequence of genes of interest is carried in the T-DNA zone of pCAM1305, comprises starch branch enzyme gene sbeIIb, marker gene glucuronidase gene GUS and the anti-hygromycin gene HPT of screening-gene.Starch branch enzyme gene sbeIIb sheet segment length 643bp constitutes the expression framework jointly with corn promotor Ubi-1,35S and no terminator.Bacterial strain is that EHA10 conveniently buys or give acquisition, in general plant genetic laboratory preservation is arranged all, referring to document Wu Guan front yard etc., and Scientia Agricultura Sinica, 2005,38 (12): 2395-2402.
Among the present invention; The histochemical stain method of said marker gene glucuronidase gene GUS is referring to Rueb and Hensgens. Rice Genetics Newsletters, 1989, (6): 168-169; The molecular detecting method of the anti-hygromycin gene HPT of screening-gene is referring to document Wu Guan front yard etc.; Scientia Agricultura Sinica, 2005,38 (12): 2395-2402.
Among the present invention, the assay method of said starch gel performance is referring to document Du pioneer etc., EI, 2001,17 (2): 16-19.
Above-mentioned stability be meant resistance starch content in the selected paddy rice between different year between (more than 2 years), season the content of (more than 2 seasons) and intersite (2 more than the place) not remarkable.
The special-purpose transgenic paddy rice of starch matrix of the present invention has the following advantages:
The present invention at first utilizes the starch retrogradation characteristic of starch branch enzyme gene sbeIIb specific aim optimization regulation and control amylose content 10% ~ 15% paddy rice.The starch of this transgenic paddy rice; Has good gel characteristic; Different fully with the starch gel resistance of the common varieties of starch based crops such as common rice, corn, wheat, can be used as novel biological culture matrix, be widely used in scientific research, food, medicine and chemical industry.
Description of drawings
Fig. 1 is the vector construction sketch map of the used genes of interest insetion sequence of the present invention.
Embodiment
Below further specify the present invention through instantiation.
Embodiment 1:
Mature embryo with amylose content 13.6% rice varieties " good educate 948 " is an explant, is seeded to inducing culture, under 25 ± 1 ℃, light intensity 3000 lux optical condition, induces the formation callus;
With inducing the callus that formed for 2 weeks, be transferred to subculture medium, successive transfer culture to callus gets into breeds the phase at double;
With the callus of propagation at double, under 25 ± 1 ℃, the dark condition of lucifuge, carry out the preparatory cultivation 2 days of acceptor callus;
To pass through pre-incubated callus (about 500 culture dishes contain the callus more than 2000), in agrobacterium suspension, soak 10 minutes; Discard bacterium liquid afterwards; Blot with aseptic filter paper, be inoculated on the common medium, under 25 ± 1 ℃, the dark condition of lucifuge, cultivated altogether 2 days;
Get above-mentioned cultured calli altogether; After cleaning, drying, transfer to screening culture medium, under 25 ± 1 ℃, the dark condition of lucifuge, cultivated for 6 weeks; Collect the resistant calli (about 210 callus) that newly grows; Be transferred on the new screening culture medium, carry out continuous 2 again and take turns the subculture screening, whenever continue 1 time at a distance from 3 turnovers;
Get 3 resistant callis (about 50 callus) of taking turns after the screening; Be transferred to differential medium; Induce seedling differentiation 78 strains; The antagonism plant carries out marker gene GUS histochemical stain and screening-gene HPT Molecular Detection, selects and remain to contain transfer-gen plant 56 strains of genes of interest starch branch enzyme gene sbeIIb insetion sequence, and the vector construction of genes of interest insetion sequence is as shown in Figure 1;
Be taken into the transfer-gen plant of choosing, gather in the crops seed behind the field planting, measure the starch gel performance, can form transfer-gen plant 11 strains of semisolid gel after the starch gelatinization of the selecting and remain cooling;
Continuous 3 years of 2008-2010; Coagulate resistance expression stability in Zhejiang Hangzhou (spring), Fuyang, Zhejiang (summer), Lingshui, Hainan (winter) three season of three locations evaluation starch respectively; Final 3 of the starch matrix rice specials of cultivating; T-NJ-1, T-NJ-2, T-NJ-3 can form stable semisolid gel after its starch gelatinization cooling, and former amylose content rice varieties " is praised and educated 948 " and then can't form semi-solid gel at all.
Claims (7)
1. the transgenic culturing method of a starch matrix rice special may further comprise the steps:
1) cut-off chain content of starch is an explant between the mature embryo of 10% ~ 15% rice varieties, is seeded to inducing culture, under 25 ± 1 ℃, light intensity 3000 lux optical condition, induces the formation callus;
2) will induce the callus that formed for 2 weeks, and be transferred to subculture medium, successive transfer culture to callus gets into breeds the phase at double;
3) callus that will breed at double carries out the preparatory cultivation 2 days of acceptor callus under 25 ± 1 ℃, the dark condition of lucifuge;
4) will pass through pre-incubated callus, in agrobacterium suspension, soak 10 minutes, discard bacterium liquid afterwards, blot, be inoculated on the common medium, under 25 ± 1 ℃, the dark condition of lucifuge, cultivate altogether 2 days with aseptic filter paper;
5) with above-mentioned cultured calli altogether, after cleaning, drying, transfer to screening culture medium; Under 25 ± 1 ℃, the dark condition of lucifuge, cultivated for 6 weeks, collect the resistant calli that newly grows, be transferred on the new screening culture medium; Carry out continuous 2 again and take turns the subculture screening, whenever continue 1 time at a distance from 3 turnovers;
6) get 3 resistant callis of taking turns after the screening; Be transferred to differential medium; Under 25 ± 1 ℃, light intensity 3000 lux optical condition, induce seedling differentiation; The antagonism plant carries out histochemical stain and Molecular Detection; Selecting and remain contains the transfer-gen plant of genes of interest insetion sequence, and said genes of interest insetion sequence is meant the insetion sequence that comprises genes of interest starch branch enzyme gene sbeIIb, marker gene glucuronidase gene GUS and the anti-hygromycin gene HPT of screening-gene, genes of interest starch branch enzyme gene sbeIIb sheet segment length 643bp;
7) be taken into the transfer-gen plant of choosing, gather in the crops seed behind the field planting, identify the starch gel performance, can form the transfer-gen plant of semisolid gel after the starch gelatinization of the selecting and remain cooling;
8) continue the transgenic line that the plantation step 7) is screened, the starch gel resistance expression stability of evaluation, the elite plant strain stable to the starch gel resistance carries out seminal propagation, is the starch matrix rice special of cultivating.
2. the breeding method of high resistant starch content paddy rice according to claim 1 is characterized in that said inducing culture adds 2 for the N6 minimal medium, 4-D1.5mg, sucrose 30g and agar powder 6.8g, pH sterilization preceding 5.8.
3. the breeding method of high resistant starch content paddy rice according to claim 1 is characterized in that said subculture medium adds 2 for the N6 minimal medium, 4-D 1mg sucrose 30g and agar powder 6.8g, pH sterilization preceding 5.8.
4. the breeding method of high resistant starch content paddy rice according to claim 1 is characterized in that said medium altogether adds 2 for the N6 minimal medium, 4-D 0.5mg, acetosyringone 1mg, sucrose 30g and agar powder 6.8g, pH sterilization preceding 5.8.
5. the breeding method of high resistant starch content paddy rice according to claim 1; It is characterized in that said screening culture medium is N6 minimal medium interpolation 2; 4-D 0.5mg, hygromycin 30mg, carboxylic Bian 300 mg, sucrose 30g and agar powder 6.8g, pH sterilization preceding 5.8.
6. the breeding method of high resistant starch content paddy rice according to claim 1; It is characterized in that said differential medium adds 6-BA1mg, NAA0.5mg, KT0.5mg, carboxylic Bian 200mg, sucrose 30g and agar powder 6.8g for the N6 minimal medium, pH sterilization preceding 5.8.
7. according to the breeding method of the described high resistant starch content paddy rice of claim 2-6, it is characterized in that said N6 minimal medium is KNO
32830mg, (NH
4)
2SO
4463mg, CaCl
22H
2O 166mg, MgSO
47H
2O 185mg, KH
2P0
4400mg, MnSO
44H
2O 4.4mg, ZnSO
47H
2O 1.5mg, FeSO
47H
2O 27.8mg, Na
2-EDTA2H2O 37.3mg, H
3BO
31.6mg, inositol 100mg, nicotinic acid 0.5mg, vitamin B6 0.5mg, vitamin B1 0.5mg and glycine 2mg.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2012100051019A CN102550414A (en) | 2012-01-10 | 2012-01-10 | Transgenic cultivation method of rice dedicated for starch medium |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2012100051019A CN102550414A (en) | 2012-01-10 | 2012-01-10 | Transgenic cultivation method of rice dedicated for starch medium |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN102550414A true CN102550414A (en) | 2012-07-11 |
Family
ID=46398071
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2012100051019A Pending CN102550414A (en) | 2012-01-10 | 2012-01-10 | Transgenic cultivation method of rice dedicated for starch medium |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102550414A (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102816778A (en) * | 2012-07-30 | 2012-12-12 | 上海市农业科学院 | Mutant gene of rice starch branching enzyme SBE3 gene and application of mutant gene |
| CN103194486A (en) * | 2013-04-18 | 2013-07-10 | 天津大学 | Hybrid poplar agrobacterium transformation method taking callus tissue as explant |
| CN104823841A (en) * | 2015-05-28 | 2015-08-12 | 浙江大学 | Method for breeding two-line hybrid rice sterile line special for starch matrix |
| CN104885926A (en) * | 2015-05-28 | 2015-09-09 | 浙江大学 | Breeding method for special three-line hybrid rice sterile line of starch substrate |
| CN106234213A (en) * | 2016-07-14 | 2016-12-21 | 浙江大学 | A kind of culture medium and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1886507A (en) * | 2003-10-27 | 2006-12-27 | 联邦科技产业研究组织 | Rice and products thereof having starch with an increased proportion of amylose |
-
2012
- 2012-01-10 CN CN2012100051019A patent/CN102550414A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1886507A (en) * | 2003-10-27 | 2006-12-27 | 联邦科技产业研究组织 | Rice and products thereof having starch with an increased proportion of amylose |
Non-Patent Citations (2)
| Title |
|---|
| 刘玉汇等: "植物淀粉分支酶基因的研究进展", 《麦类作物学报》, vol. 30, no. 3, 31 December 2010 (2010-12-31), pages 581 - 586 * |
| 朱昌兰等: "水稻低直链淀粉含量基因育种利用的研究进展", 《中国农业科学》, vol. 37, no. 2, 31 December 2004 (2004-12-31), pages 157 - 162 * |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102816778A (en) * | 2012-07-30 | 2012-12-12 | 上海市农业科学院 | Mutant gene of rice starch branching enzyme SBE3 gene and application of mutant gene |
| CN102816778B (en) * | 2012-07-30 | 2014-09-03 | 上海市农业科学院 | Mutant gene of rice starch branching enzyme SBE3 gene and application of mutant gene |
| CN103194486A (en) * | 2013-04-18 | 2013-07-10 | 天津大学 | Hybrid poplar agrobacterium transformation method taking callus tissue as explant |
| CN104823841A (en) * | 2015-05-28 | 2015-08-12 | 浙江大学 | Method for breeding two-line hybrid rice sterile line special for starch matrix |
| CN104885926A (en) * | 2015-05-28 | 2015-09-09 | 浙江大学 | Breeding method for special three-line hybrid rice sterile line of starch substrate |
| CN104885926B (en) * | 2015-05-28 | 2019-02-05 | 浙江大学 | A kind of dedicated series of three-series hybrid rice sterile line breeding method of starch matrix |
| CN104823841B (en) * | 2015-05-28 | 2019-02-05 | 浙江大学 | A kind of dedicated double-linear hybrid rice sterile line breeding method of starch matrix |
| CN106234213A (en) * | 2016-07-14 | 2016-12-21 | 浙江大学 | A kind of culture medium and application thereof |
| CN106234213B (en) * | 2016-07-14 | 2018-02-23 | 浙江大学 | A kind of culture medium and its application |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Joshi et al. | In vitro screening of rice genotypes for drought tolerance using polyethylene glycol | |
| CN107312793A (en) | The tomato dna editor carrier of Cas9 mediations and its application | |
| CN103215280B (en) | Peanut SPL (squamosa promoter-binding protein-like) transcription factor gene, as well as encoded protein and application thereof | |
| CN102550414A (en) | Transgenic cultivation method of rice dedicated for starch medium | |
| CN104542256A (en) | Breeding method for importing false smut resistance of indica rice into japonica rice | |
| CN104004781A (en) | Preparation method of glyphosate resistant transgenic rice | |
| CN103966235B (en) | Eggplant anthocyanidin synthesis related gene SmMYB1 and recombinant expression vector thereof and cultivating the application in purple solanum lycopersicum | |
| CN104429932A (en) | Novel indica-japonica hybrid gene introgression sheath blight resistance breeding method | |
| CN105191794B (en) | Method for rapid propagation of bischofia javanica based on embryogenic callus seedling development | |
| CN101006768A (en) | Agrobacterium-mediated Sophora japonica transgenic and tissue-culturing rapid propagation method | |
| CN101643746B (en) | Hotness-resistant Lucerne transgenic culturing method | |
| CN106234208B (en) | A kind of fast breeding method of odor type top grade high-grade rice | |
| CN102577950A (en) | Cultivating method of indigofera pseudotinctoria mats with high flavone content | |
| CN104450743A (en) | Application of eggplant anthocyanidin synthesis associated gene SmMYB1 in cultivation of solanum aethiopicum group gilo variety | |
| CN104285792B (en) | A kind of tissue rapid propagation method of Alnus formosana Plantation | |
| CN102577948A (en) | Rice cultivating method capable of improving resistant starch content | |
| CN101775409A (en) | Method for obtaining transgenic corns by infecting young ears with agrobacterium | |
| CN102102108B (en) | Method for cultivating efficient selected-marker-free transgenic crop by using double T-DNA+1 vectors | |
| CN104726488A (en) | Method for culturing stress-resistance herbicide-resistance transgenic aerobic rice | |
| CN102550415A (en) | Method for cultivating rice with high resistant starch content | |
| CN108795927A (en) | The clone of common wheat gene TaSPX3 coded sequences and its application | |
| CN102524075A (en) | Method for culturing chrysanthemum morifolium ramat with high flavones content | |
| CN104560961A (en) | Molecular markers linked to ZYMV (zucchini yellow mosaic virus-1) dominant disease-resistant gene ZYMV-1 and application of molecular markers | |
| CN106754970A (en) | A kind of method for cultivating the type of resistance to bolting romaine lettuce by controlling LsFT genes | |
| CN106171960B (en) | A kind of selection of fringe portion without wax malting barley variety |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20120711 |