CN102304506A - Plant transgenic selection system using betaine aldehyde dehydrogenase (BADH) as marker gene and use thereof - Google Patents
Plant transgenic selection system using betaine aldehyde dehydrogenase (BADH) as marker gene and use thereof Download PDFInfo
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Abstract
The invention discloses a plant transgenic selection system using betaine aldehyde dehydrogenase (BADH) as a marker gene and use thereof and belongs to the technical field of gene engineering. In the system, BADH is used as a marker gene of an exogenous gene and NaCl is used as a selecting agent. The use comprises the following steps: (1) determining NaCl concentration which can just inhibit plant tissue differentiation; (2) transferring the exogenous gene which contains the BADH marker gene into a plant cell or tissue; and (3) culturing the transgenic tissue or cell into which the BADH marker gene is transferred on a culture medium in which the NaCl concentration determined by the step (1), obtaining the transgenic tissue or cell into which the BADH marker gene is transferred into if the transgenic tissue or cell can grow, and confirming by molecular detection. The invention has the advantages of greatly lowering the cost investment in a transgenic test, solving the safety problem of transgenic organisms, and providing low cost selection for the construction of a safe and effective selection system for plant genetic transformation.
Description
Technical field:
The invention belongs to gene engineering technology field, be specifically related to serve as a mark the plant transgene selective system and the application thereof of gene with BADH.
Background technology:
The utilization genetic engineering technique realizes that the genetic improvement of plant trait has become a kind of new trend of the new variety that cultivate plants.In the plant transgene process, the employing of effective choice system is a key link of plant transgene success or not.The most frequently used selective system is weedicide selective system and microbiotic selective system at present; Although these two kinds of selective systems have been successfully used to the genetic transformation of various plants, the employing of these two kinds of selective systems is to cause the major reason of the public to the genetically modified organism safety concerns.On the one hand, the antibiotics resistance gene in fears are entertained that the transgenic plant can be transferred in the environmental microorganism, and the result can cause the increase of resistance pathogenic agent; On the other hand, anti-herbicide gene may be passed to ruderal, thereby makes weeds obtain Herbicid resistant.Though these worry also not have scientific basis; But the public can have a strong impact on the development of plant genetic engineering to the worry of the potential hazard of selectable marker gene; So; Set up the selective system of plant genetic conversion safely and effectively, the transgenic plant of cultivating the non-resistant marker gene have significance on genetic engineering breeding.Current, the resistant maker gene safety issue has two kinds of approach in the solution transgenic plant: still use resistant maker gene when (1) transforms, behind the transgenic plant reproductive success, before discharging the land for growing field crops, resistant maker gene is rejected; (2) development security sign gene be used for plant genetic transform (Zhao Yan, Wang Huizhong, Yu Yanchun etc. the security New Policy [J] of marker gene in the transgenic plant. heredity, 2003,25 (1): 119-122).Since before a kind of approach complicated operation, be unfavorable for that the quick commercialization of transgenic plant is used, therefore in the plant transgene operation, tend to adopt the genetic transformation method of security sign gene at present more.
Betaine-aldehyde dehydrogenase (BADH) gene is the key enzyme in the trimethyl-glycine pathways metabolism, and this enzyme can (have another name called phytotoxic betaine aldehyde chloride: formyl methyl trimethoxy base ammonia chloride, English name: Betaine aldehyde chloride; Molecular formula: C5H12C1NO) being converted into favourable trimethyl-glycine, is the security gene of generally acknowledging in the world, but and double as marker gene use.Many transgenosis achievements show that the importing of external source BADH gene can improve the ability of phytosynthesis trimethyl-glycine; And (Sakamoto A such as drought resistance of enhancement of plant, salt tolerance; Murata N.The role of glycine betaine in protection of plants from stress clues from transgenic plants[J] .Plant Cell and Evironment; 2002,25:163-171; Pay light, Su Qiao, Wu Wei etc. change the acquisition and the salt tolerance [J] thereof of BADH gene corn. gas, 2006,29 (3): 344-347) are learned by Liaoning Normal University.At present; With betaine aldehyde dehydrogenase gene double as marker gene at tobacco (H Daniell; B Muthukumar; S B Lee.Marker free transgenic plants:engineeringthe chloroplast genome without the use of antibiotic selection[J] .Current Genetics; 2001; 39 (2): 109-116), two look Stem or leaf of Shrub Lespedeza (Study on Genetic Transformation [D] of Yang Xiaohong .BADH gene, antisense 4CL gene pairs two look Stem or leaf of Shrub Lespedeza. Beijing Forestry University's doctorate paper, 2009) on obtained success.Although this genetic transformation technology has been proved to be feasible; But this kind method for transformation is to provide with betaine aldehyde chloride to select to press; Betaine aldehyde chloride then is the extremely expensive selective agent of a kind of price; The world market valency up to 1000 dollars/gram about; Domestic price also reaches 2500～3000 yuan/gram; Cause this kind genetic transformation cost high, and then limited being widely used of this genetic transforming method.Therefore, explore low-cost genetic transforming method, to promoting being widely used of this security genetic transforming method significant with the quick commercialization application of transgenosis achievement with betaine aldehyde dehydrogenase gene double as marker gene.
Summary of the invention:
NaCl is kind of the compound to plant tissue, cell development generation murder by poisoning, the Na of its generation
+A large amount of accumulation can destroy in the cell ionic equilibrium and suppress Physiological and Biochemical Metabolism process in the cell in cell, make the photosynthesis of plant ability drop, and are finally hungry dead because of carbon.Plant then can not carry out normal tissue differentiation containing on the certain density NaCl substratum, and when NaCl concentration was high, plant can be dead.But NaCl is cheap, and just 6～8 yuans of per 500 grams are easy to buy on market.With BADH gene double as selectable marker gene the time; Provide selection to press by NaCl; With this compound with low cost as selective agent; To intending the plant transformed explant; Be placed on earlier on the NaCl substratum that contains different concns; Observe the influence degree of NaCl, find and just in time can suppress the NaCl concentration that plant tissue breaks up the plant tissue differentiation, with this concentration as NaCl selective agent concentration in the plant genetic conversion process.Have the advantages that to improve salt tolerance owing to change the plant tissue or the cell of BADH gene over to; Therefore; Containing on the certain density NaCl substratum; Changeing the cell or tissue of BADH gene can normal differentiation grow; Non-transformed cell or tissue can not break up; Thereby can obtain to have the resistant buds of NaCl resistance, these resistant budses can be used as the plant original material that changes foreign gene over to.After, these resistant budses are further grown containing on the certain density NaCl substratum, and it is carried out Molecular Detection, just can obtain transfer-gen plant with the double gene that makes marks of BADH gene.
Present in order to solve in the marker gene that adopts BADH as foreign gene, when providing selection to press with betaine aldehyde chloride; The high problem of genetic transformation cost that is caused, the invention provides a kind of when serving as a mark gene with BADH the plant transgene selective system and the application of this plant transgene selective system is provided simultaneously.
With the serve as a mark plant transgene selective system of gene of BADH, comprise foreign gene and selective agent, wherein, contain BADH in the said foreign gene as selectable marker gene, said selective agent is NaCl.
The described plant transgene selective system of technique scheme, wherein, said plant is two look Stem or leaf of Shrub Lespedeza or tobaccos.
Described in the technique scheme with the serve as a mark application of plant transgene selective system in transgenic plant genetic engineering of gene of BADH; Comprise through transgenic method and will import in the plant as the foreign gene of selectable marker gene with BADH; Wherein, also comprise the steps:
(1), add the NaCl of different concns in the tissue of the plant before transgenosis or the cell culture medium, confirm just in time can suppress the NaCl concentration that plant tissue or cell break up;
(2), the tissue with NaCl resistance that possibly transform foreign gene that will obtain or cell be placed on the determined NaCl concentration of step (1) substratum on cultivate, being of can growing changed the material of BADH as the foreign gene of selectable marker gene over to.
Application described in the technique scheme, wherein, with the serve as a mark application of plant transgene selective system in two look Stem or leaf of Shrub Lespedeza or tobacco transgenic engineering of gene of BADH.
Application described in the technique scheme comprises through transgenic method will being that the foreign gene of marker gene changes the step in the two look Stem or leaf of Shrub Lespedeza over to BADH, wherein, also comprises the steps:
(1), in the substratum of the first two look Stem or leaf of Shrub Lespedeza cotyledonary node differentiation adventitious buds of transgenosis, add the NaCl of different concns, confirm just in time can suppress the NaCl concentration of two look Stem or leaf of Shrub Lespedeza cotyledonary node differentiation adventitious buds;
(2), the above-mentioned two look Stem or leaf of Shrub Lespedeza cotyledonary nodes that changed foreign gene over to are placed the same medium that contains in steps the NaCl concentration of (1) confirming select;
(3), in the first two the look Stem or leaf of Shrub Lespedeza adventitious bud proliferation of transgenosis and the substratum of taking root, add the NaCl of different concns, confirm just in time can suppress two look Stem or leaf of Shrub Lespedeza adventitious bud proliferations and the NaCl concentration of taking root;
(4), the salt tolerant resistant buds that obtains in the step (2) is carried out selective proliferative and takes root in the substratum of the determined NaCl concentration of step (3), to strengthen selecting effect.
Application described in the technique scheme comprises through transgenic method will being that the foreign gene of marker gene changes the step in the tobacco leaf over to BADH, wherein, also comprises the steps:
(1), before transgenosis, add the NaCl of different concns in the substratum of tobacco leaf differentiation adventitious buds, confirm just in time can suppress the NaCl concentration of tobacco leaf differentiation adventitious buds;
(2), the tobacco leaf that will change foreign gene over to places the same medium that contains in steps NaCl concentration of (1) confirming to select;
(3), add the NaCl of different concns in tobacco stem section propagation and tissue cultured seedling are taken root before transgenosis the substratum, confirm just in time to suppress the NaCl concentration that tobacco stem section propagation and tissue cultured seedling are taken root;
(4), the Tobacco Salt resistant buds that obtains in the step (2) is carried out selectivity stem section propagation take root in the substratum of the determined NaCl concentration of step (3), to strengthen the selection effect with tissue cultured seedling.
The described application of arbitrary technical scheme in the technique scheme wherein, is also comprising the step of confirming through Molecular Detection through containing the NaCl substratum after selecting; Said Molecular Detection is preferably the PCR detection or Southern detects.
The present invention has following beneficial effect:
1, because the present invention adopts NaCl as selective agent, compare with betaine aldehyde chloride commonly used now, greatly reduce the cost input in the transgenosis test;
2, when adopting NaCl as selective agent; With the BADH gene is that marker gene is when carrying out the foreign gene genetic transformation; Betaine aldehyde dehydrogenase gene is used in transgenic engineering on a large scale; Solved the genetically modified organism safety issue that present employing weedicide selective system and microbiotic selective system are caused, for the selective system of setting up plant genetic conversion safely and effectively provides possibility cheaply;
3, with NaCl as selective agent, with BADH gene double as marker gene plant is carried out genetic transformation, the gained transgenic plant are very fast easily through national transgenosis safety evaluation, carry out commercial production.
Description of drawings:
1, Fig. 1 is that the first two look Stem or leaf of Shrub Lespedeza cotyledonary node of transgenosis is on the substratum that does not contain NaCl;
2, Fig. 2 is that the first two look Stem or leaf of Shrub Lespedeza cotyledonary node of transgenosis is on the substratum that contains 0.8g/L NaCl;
3, Fig. 3 is the stem section propagation of the first two look Stem or leaf of Shrub Lespedeza of transgenosis on the substratum that does not contain NaCl;
4, Fig. 4 is the propagation performance of the first two look Stem or leaf of Shrub Lespedeza of transgenosis on the substratum that contains 0.8g/L NaCl;
5, Fig. 5 is taken root on the substratum that does not contain NaCl for the first two look Stem or leaf of Shrub Lespedeza of transgenosis;
6, Fig. 6 is the take root performance of the first two look Stem or leaf of Shrub Lespedeza of transgenosis on the substratum that contains 0.5g/L NaCl;
7, Fig. 7 is transgenosis two look Stem or leaf of Shrub Lespedeza cotyledonary node resistant budses on the NaCl selective agent;
8, Fig. 8 is two look Stem or leaf of Shrub Lespedeza transformed plant PCR detected results;
9, Fig. 9 is two look Stem or leaf of Shrub Lespedeza transformed plant Southern detected results;
10, Figure 10 breaks up on the substratum that does not contain NaCl for tobacco leaf before the transgenosis;
11, Figure 11 is tobacco leaf differentiation situation on the substratum of the NaCl that contains 2g/L before the transgenosis;
12, Figure 12 breeds in the substratum that does not contain NaCl for tobacco stem section before the transgenosis and takes root;
13, Figure 13 is tobacco stem section performance situation on the substratum of the NaCl that contains 2g/L before the transgenosis;
14, Figure 14 is the transgene tobacco resistant buds on the NaCl selective agent;
15, Figure 15 is a tobacco transformed plant PCR detected result;
16, Figure 16 is a tobacco transformed plant Southern detected result;
17, Figure 17 is the propagation performance of two look Stem or leaf of Shrub Lespedeza after the transgene on the substratum that contains 1.0g/L NaCl;
18, Figure 18 is the take root performance of two look Stem or leaf of Shrub Lespedeza on the substratum that contains 1.0g/L NaCl after the transgene;
19, Figure 19 shows on the substratum of the NaCl that contains 3g/L for the tobacco after the transgene.
Embodiment:
For making technical scheme of the present invention be convenient to understand, the present invention is further illustrated below in conjunction with Figure of description and specific embodiment.
The test operation of the genetic transformation of plant transgene part, foreign gene part also can be operated according to the method described in the cited paper according to well known to a person skilled in the art that working method operates in following examples.
Embodiment 1:With the double gene that makes marks of BADH gene, as selective agent two look Stem or leaf of Shrub Lespedeza are carried out the genetic transformation process with NaCl
One; Confirm to suppress the NaCl concentration of two look Stem or leaf of Shrub Lespedeza cotyledonary node differentiation adventitious buds: the explant that adopts according to two look Stem or leaf of Shrub Lespedeza genetic transformation processes is a cotyledonary node; Cotyledonary node differentiation adventitious buds effect on the substratum of MS+6-BA 0.2mg/L+3% sucrose+6g/L agar is best; Be the basis with this substratum; Configuration contains the substratum of different concns NaCl; As: MS+6-BA0.2mg/L+3% sucrose+6g/L agar+NaCl 0.4g/L; MS+6-BA 0.2mg/L+3% sucrose+6g/L agar+NaCl0.6g/L; MS+6-BA 0.2mg/L+3% sucrose+6g/L agar+NaCl 0.8g/L; MS+6-BA 0.2mg/L+3% sucrose+6g/L agar+serial substratum such as NaCl 1.0g/L; Observe the differentiation adventitious buds situation of cotyledonary node on these substratum; Because two look Stem or leaf of Shrub Lespedeza cotyledonary nodes can not differentiate indefinite bud on MS+6-BA 0.2mg/L+3% sucrose+6g/L agar+NaCl 0.8g/L substratum; Therefore, think and add 0.8gL in the substratum
-1NaCl just can suppress differentiation adventitious buds, and this concentration is exactly the selection pressure when as selective agent two look Stem or leaf of Shrub Lespedeza being carried out genetic transformation with NaCl.
On the substratum of the best MS+6-BA 0.2mg/L+3% sucrose+6g/L agar of cotyledonary node differentiation adventitious buds effect, add 0.8gL
-1NaCl obtains selecting substratum, will be that two look Stem or leaf of Shrub Lespedeza cotyledonary nodes after the foreign gene of marker gene transforms are placed on and select to screen in the substratum with BADH;
Fig. 1 shows on the substratum that does not contain NaCl for the first two look Stem or leaf of Shrub Lespedeza cotyledonary node of transgenosis; Fig. 2 shows on the substratum that contains 0.8g/L NaCl for the first two look Stem or leaf of Shrub Lespedeza cotyledonary node of transgenosis, and Fig. 7 is transgenosis two look Stem or leaf of Shrub Lespedeza cotyledonary node resistant budses on the NaCl selective agent.
Two, understand NaCl concentration to the two look Stem or leaf of Shrub Lespedeza adventitious bud proliferations and the influence of taking root: two look Stem or leaf of Shrub Lespedeza adventitious bud proliferation suitable culture bases are MS+6-BA 1.0mg/L+NAA 0.1mg/L, and root media is: MS+IBA 0.4mg/L+NAA0.2mg/L.On the basis of these media with different concentrations of NaCl, observed adventitious buds or roots, and found adventitious buds containing 0.8g · L
-1 NaCl not proliferate on media , containing 0.5g · L
-1 NaCl rooting medium can not, therefore, determine the medium added 0.8g · L
-1 NaCl as resistant shoots obtained by genetic transformation of value-added in the process of selection pressure, medium supplemented with 0.5g · L
-1 NaCl as bicolor resistance Bud rooting process selection pressure, Figure 3 shows the transformation of former bicolor medium in the absence of NaCl on the stem proliferation, Figure 5 is transformed before bicolor in the absence of NaCl, rooting, Figure 4 is a converted former bicolor containing 0.8g / L? NaCl medium on the proliferation of performance, Figure 6 is converted former bicolor containing 0.5g / L? NaCl on the rooting medium performance Figure 8 is a bicolor transgenic PCR test, Figure 9 is a bicolor transgenic Southern blot analysis, in which Figures 8 and 9, ck as positive control (i.e. containing the foreign gene of plasmid DNA), ck - as a negative control (ie, non-transgenic plants), B2? B3? B4? B5 represents gene transfer occurs after four in NaCl strains grown on selective agent, after PCR testing, there are bands B3, B5 strains is the transgenic plants.Figure 17 is the stem section increment performance of two look Stem or leaf of Shrub Lespedeza after the transgene on the substratum that contains 1.0g/L NaCl; Figure 18 is taken root on the substratum that contains 1.0g/L NaCl for two look Stem or leaf of Shrub Lespedeza after the transgene.
Embodiment 2:With the double gene that makes marks of BADH gene, as selective agent tobacco is carried out genetic transformation with NaCl:
One, understand the influence of NaCl concentration to tobacco leaf differentiation adventitious buds ability: the appropriate media of tobacco leaf differentiation is 1/2MS+6-BA 2.0mg/L+NAA 0.1mg/L+3% sucrose+6g/L agar.On the basis of this substratum; Add 0,0.4,0.8,1.2,1.6,2.0 respectively, the NaCl of 3.0g/L; Observe the growth performance of tobacco leaf on various substratum, confirm in substratum, to add 2g/L NaCl and just can suppress tobacco leaf differentiation indefinite bud;
On the appropriate media 1/2MS+6-BA 2.0mg/L+NAA 0.1mg/L+3% sucrose+6g/L agar of tobacco leaf differentiation, add 2gL
-1NaCl obtains selecting substratum, will be that tobacco leaf after the foreign gene of marker gene transforms is placed on and selects to screen in the substratum with BADH;
Figure 10 breaks up on the substratum that does not contain NaCl for tobacco leaf before the transgenosis, and Figure 11 is the preceding tobacco leaf differentiation situation on the substratum of the NaCl that contains 2g/L of transgenosis, and Figure 14 is the transgene tobacco blade resistant buds on the NaCl selective agent;
Two, understand the influence of NaCl concentration to tobacco stem section multiplication capacity, tissue cultured seedling rootability: the appropriate media of tobacco stem section propagation is 1/2MS+6-BA 1.0mg/L+NAA 0.1mg/L+3% sucrose+6g/L agar; The appropriate media of taking root is 1/2MS+IAA0.5mg/L+3% sucrose+6g/L agar.Respectively preparation be added with 0.4,0.8,1.2,1.6,2.0, the stem section of the NaCl of 3.0g/L propagation, root media, the NaCl that confirms in substratum, to add 2g/L just can suppress the further differentiation and proliferation of tobacco stem section and tissue cultured seedling is taken root.Therefore; NaCl to add 2g/L in the culture medium presses as the selection in selection pressure in the tobacco leaf genetic transformation and the resistant buds propagation rooting process; Figure 12 breeds in the culture medium that does not contain NaCl for tobacco stem section before the transgenosis and takes root; Figure 13 is tobacco stem section performance situation on the culture medium of the NaCl that contains 2mg/L before the transgenosis; Figure 15 is a tobacco transformed plant PCR testing result; Figure 16 is a tobacco transformed plant Southern testing result; The positive contrast of ck+ in Figure 15 and Figure 16; The negative contrast of ck- (being the non-transgenic strain); On behalf of gene, Y2Y3Y4Y6Y8 change the strain system numbering that back 5 energy of appearance are grown on the NaCl selective agent; Detect through PCR, have the Y4 of band, Y6 strain system to be only transfer-gen plant.Figure 19 changes tobacco plant afterwards in the performance that contains on the NaCl substratum of 3g/L for gene.
The above; It only is preferred embodiment of the present invention; Be not that the present invention is done any formal and substantial restriction; Allly be familiar with the professional and technical personnel; In not breaking away from technical scheme scope of the present invention; When can utilizing the above technology contents that discloses, and a little change of making, modify the equivalent variations with differentiation, be equivalent embodiment of the present invention; Simultaneously, all foundations essence technology of the present invention all still belongs in the scope of technical scheme of the present invention change, modification and the differentiation of any equivalent variations that above embodiment did.
Claims (8)
1. with the serve as a mark plant transgene selective system of gene of BADH, comprise foreign gene and selective agent, it is characterized in that: contain the BADH as selectable marker gene in the said foreign gene, said selective agent is NaCl.
2. plant transgene selective system according to claim 1 is characterized in that: said plant is two look Stem or leaf of Shrub Lespedeza or tobaccos.
3. claim 1 is described with the serve as a mark application of plant transgene selective system in transgenic plant genetic engineering of gene of BADH; Comprise through transgenic method will comprising in the foreign gene importing plant of BADH as selectable marker gene, it is characterized in that: also comprise the steps:
(1), add the NaCl of different concns in the tissue of the plant before transgenosis or the cell culture medium, confirm just in time can suppress the NaCl concentration that plant tissue or cell break up;
(2), the tissue with NaCl resistance that possibly transform foreign gene that will obtain or cell be placed on the determined NaCl concentration of step (1) substratum on cultivate, being of can growing changed the material of BADH as the foreign gene of selectable marker gene over to.
4. application according to claim 3 is characterized in that: with the serve as a mark application of plant transgene selective system in two look Stem or leaf of Shrub Lespedeza or tobacco transgenic engineering of gene of BADH.
5. application according to claim 4 comprises that will comprise the serve as a mark foreign gene of gene of BADH through transgenic method changes the step in the two look Stem or leaf of Shrub Lespedeza over to, is characterized in that: also comprise the steps:
(1), in the substratum of the first two look Stem or leaf of Shrub Lespedeza cotyledonary node differentiation adventitious buds of transgenosis, add the NaCl of different concns, confirm just in time can suppress the NaCl concentration of two look Stem or leaf of Shrub Lespedeza cotyledonary node differentiation adventitious buds;
(2), the above-mentioned two look Stem or leaf of Shrub Lespedeza cotyledonary nodes that changed foreign gene over to are placed the same medium that contains in steps the NaCl concentration of (1) confirming select;
(3), in the first two the look Stem or leaf of Shrub Lespedeza adventitious bud proliferation of transgenosis and the substratum of taking root, add the NaCl of different concns, confirm just in time can suppress two look Stem or leaf of Shrub Lespedeza adventitious bud proliferations and the NaCl concentration of taking root;
(4), the salt tolerant resistant buds that obtains in the step (2) is carried out selective proliferative and takes root in the substratum of the determined NaCl concentration of step (3), to strengthen selecting effect.
6. application according to claim 4 comprises that will comprise the serve as a mark foreign gene of gene of BADH through transgenic method changes the step in the tobacco leaf over to, it is characterized in that, also comprises the steps:
(1), before transgenosis, add the NaCl of different concns in the substratum of tobacco leaf differentiation adventitious buds, confirm just in time can suppress the NaCl concentration of tobacco leaf differentiation adventitious buds;
(2), the tobacco leaf that will change foreign gene over to places the same medium that contains in steps NaCl concentration of (1) confirming to select;
(3), add the NaCl of different concns in tobacco stem section propagation and tissue cultured seedling are taken root before transgenosis the substratum, confirm just in time to suppress the NaCl concentration that tobacco stem section propagation and tissue cultured seedling are taken root;
(4), the Tobacco Salt resistant buds that obtains in the step (2) is carried out selectivity stem section propagation take root in the substratum of the determined NaCl concentration of step (3), to strengthen the selection effect with tissue cultured seedling.
7. according to the described application of arbitrary claim in the claim 3～6, it is characterized in that: also comprising the step of confirming through Molecular Detection after selecting through containing the NaCl substratum.
8. application according to claim 7 is characterized in that: said Molecular Detection is that PCR detects or Southern detects.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7795497B2 (en) * | 2000-03-02 | 2010-09-14 | University Of Central Florida Research Foundation, Inc. | Marker free transgenic plants: engineering the chloroplast genome without the use of antibiotic selection |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| US7795497B2 (en) * | 2000-03-02 | 2010-09-14 | University Of Central Florida Research Foundation, Inc. | Marker free transgenic plants: engineering the chloroplast genome without the use of antibiotic selection |
Non-Patent Citations (4)
| Title |
|---|
| DANIELL H.等: "Marker free transgenic plants: engineering the chloroplast genome without the use of antibiotic selection", 《CURRENT GENETICS》, vol. 39, no. 2, 30 April 2001 (2001-04-30), pages 109 - 116, XP002979049, DOI: doi:10.1007/s002940100185 * |
| 李明等: "二色胡枝子遗传转化中抗生素浓度优化研究", 《广东农业科学》, no. 5, 31 May 2009 (2009-05-31), pages 44 - 47 * |
| 杨晓红: "BADH基因、反义4CL基因对二色胡枝子的遗传转化研究", 《中国博士学位论文全文数据库-农业科技辑》, vol. 39, no. 2, 15 October 2009 (2009-10-15) * |
| 赵艳等: "一种简便的获得无标记耐盐转基因水稻植株的NaCl有效筛选浓度选择法", 《中国水稻科学》, vol. 25, no. 3, 10 May 2011 (2011-05-10), pages 243 - 248 * |
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