CN103477980A - Young embryo callus induction and plant regeneration method for pop corns - Google Patents
Young embryo callus induction and plant regeneration method for pop corns Download PDFInfo
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- CN103477980A CN103477980A CN201310401998.1A CN201310401998A CN103477980A CN 103477980 A CN103477980 A CN 103477980A CN 201310401998 A CN201310401998 A CN 201310401998A CN 103477980 A CN103477980 A CN 103477980A
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Abstract
The invention discloses a young embryo callus induction and plant regeneration method for Yuanzihong pop corns. The method comprises the steps of inducing callus from a young embryo of a corn inbred line, subculturing twice, and selecting a corn embryonic type callus to directly generate an entire plant, wherein the corn inbred line is N10 or N04. The regeneration system is further constructed and optimized on the basis of research of the pop corns, so that a platform is provided for genetic transformation by use of the pop corn inbred line as a receptor. According to the method, a material for genetic transformation is provided, the pop corn inbred line is used as a transformation receptor, the background is homozygous, the work of backcrossing with recurrent parents for multiple times is omitted, and the phenotype of the transformed generations can be easily observed. The platform is provided for transformation of the pop corns, and the workload of backcrossing of multiple generations after transformation is alleviated. And furthermore, the pop corn plants and the pop corn kernels have unique characteristics, so that the phenotype of transformed generations can be conveniently identified.
Description
Technical field
The present invention relates to a kind of corn breeding method, be specifically related to the method for pop corn rataria callus induction and plant regeneration.
Background technology
At present, along with completing of corn inbred line B73 large scale sequencing, cloned a lot of useful genes, the development of technique for gene engineering means and being gradually improved in addition, for transgenic breeding is laid a good foundation.Since the acquisition of the first strain transfer-gen plant in 1984, the world has carried out the every research to transgenic technology, also corn is carried out the research of character improvement and gene function simultaneously.It has been one of study hotspot both domestic and external that maize genetic transforms.
Do genetically modified correlative study good transformation system will be arranged, the world today has studied a lot of to the transformation system of conventional corn, especially utilize the Study on Transformation of crossbreed HiII, but what transgenic progeny need to be carried out many generations backcrosses purifying genetic background, just can obtain the needed transgenic line of researcher.In recent years, researchers also are devoted to study the regenerating system of corn inbred line.2011, Tian Fenglong was neat 319 to corn inbred line, prosperous 7-2,18-599 and 178 rataria callus induction, and the method then infected with Agrobacterium has obtained the transfer-gen plant of DWF4.2011, Ji Lili etc. take 922, H99, red 598 and red 988 4 corn inbred lines be material, optimized the adjusting of inducing of rataria callus.2009, the rataria of Tao Fang philosophy usage 059,4K060,4K061,4K261, a 4K2965 high oil corn self interweaving series was set up and has been optimized its regenerating system.2007, Hou Lihong set up its regenerating system with the rataria of high oil corn self interweaving series GY220, GY246, GY302, GY798 and GY923 respectively.2004, it was test material that Hu Jianguang etc. be take the ratarias such as sweet No. 3 of super-sweet corn inbred line S1, S2, S3 and Guangdong, sets up its efficient strain system.2007, beam saussurea involucrata etc. be take the Glutinous maize rataria as experiment material, sets up its regenerating system, and has successfully obtained the transformed plant of cpTI and Bar gene with agriculture bacillus mediated method.And it is still blank so far to the research of pop corn regenerating system and genetic transformation thereof.The research of pop corn inbred line genetic transformation has a lot of advantage.At first, with conventional corn, compare, pop corn is shorter breeding time; Secondly, with conventional corn, compare, the pop corn plant is little, and each tree characteristics all has significant difference; And its seed is little, be convenient to observe the discrepant phenotype of tool.If the related gene of research is to control the aspects such as tree characteristics, seed size, output, the pop corn of take carries out genetic transformation as acceptor, by the investigation to each proterties of offspring, can be easy to obtain phenotypic related data, and then can realize the deep structure research to related gene.Again, with the pop corn selfing, be the genetic transformation of acceptor, screening gained T1 has phenotype for positive plant, does not need repeatedly to backcross purifying genetic background again, can analyze relatively in generation morning, time saving and energy saving.Finally, pop corn itself just has higher nutritive value, by genetic transformation, realizes high yield, is also an important research direction.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of method of pop corn rataria callus induction and plant regeneration.
Technical scheme of the present invention is: the method for pop corn rataria callus induction and plant regeneration, first the corn inbred line rataria is induced to callus, through twice subculture, choose wherein corn embryonic type callus, Direct Regeneration becomes whole plant, described corn inbred line is N10 or N04, and its step is as follows:
(1) after getting the immature ear sterilization treatment that the artificial autocopulation pollinates rear 10-11 days, allocation rataria in superclean bench, plumular axis, towards being placed down on inducing culture, induces maize calli for the first time;
(2) maize calli for the first time obtained by step (1) cuts plumule and transfers on new inducing culture, after 15 days, obtains maize calli for the second time;
(3) after the maize calli for the second time obtained by step (2) is caught broken, transfer on new inducing culture, every 15 days subcultures once, subculture twice, obtain corn embryonic type callus;
(4) the corn embryonic type callus obtained by step (3) is transferred on differential medium and is obtained the corn seedling of growing thickly;
(5) corn step (4) the obtained seedling of growing thickly is divided into single seedling, proceeds to regeneration whole plant in root media.
In described step (1), step (2), step (3), the inducing culture of N04 is: MS minimal medium 4 g/L+N6 vitamin (1000x) 1ml+2,4-D 0.4 mg/L+L-PROLINE 5 g/L+sucrose 30 g/L+inositol 0.1 g/L+caseinhydrolysate 0.1 g/L+AgNO
336 μ M/L+plant gel 3 g/L, pH is 5.8.
In described step (1), step (2), step (3), the inducing culture of N10 is: MS minimal medium 4 g/L+N6 vitamin (1000x) 1ml+2,4-D 2 mg/L+L-PROLINE 2.76 g/L+sucrose 30 g/L+inositol 0.1 g/L+caseinhydrolysate 0.1 g/L+AgNO
313 μ M/L+plant gel 3 g/L, pH is 5.8.
Differential medium in described step (4) is: MS minimal medium 4 g/L+inositol 0.1 g/L+sucrose 30 g/L+6-BA 0.2 mg/L+plant gel 3 g/L, pH is 5.8.
In described step (5), root media is: MS minimal medium 4 g/L+inositol 0.1 g/L+sucrose 30 g/L+NAA 0.2 mg/L+plant gel 30 g/L, pH is 5.8.
The invention has the beneficial effects as follows: on the basis of the present invention with regard to pop corn research, further its regenerating system is built and optimized, thereby for being that acceptor carries out genetic transformation platform is provided with the pop corn selfing.The invention provides a kind of material of genetic transformation, utilize the pop corn inbred line as transformation receptor, background is isozygotied, and dispenses the work repeatedly backcrossed with recurrent parent, and is easy to observe the phenotype of transformation generation.The present invention can provide platform for the conversion of pop corn, has alleviated and has transformed rear number for the workload backcrossed.And the pop corn plant, seed all has own unique characteristics, is convenient to the phenotypic evaluation of transformation generation.
Embodiment
The method of pop corn rataria callus induction and plant regeneration, first induce callus by the corn inbred line rataria, through twice subculture, choose wherein corn embryonic type callus, Direct Regeneration becomes whole plant, and described corn inbred line is N10 or N04, and its step is as follows:
(1) after getting the immature ear sterilization treatment that the artificial autocopulation pollinates rear 10-11 days, allocation rataria in superclean bench, plumular axis, towards being placed down on inducing culture, induces maize calli for the first time;
(2) maize calli for the first time obtained by step (1) cuts plumule and transfers on new inducing culture, after 15 days, obtains maize calli for the second time;
(3) after the maize calli for the second time obtained by step (2) is caught broken, transfer on new inducing culture, every 15 days subcultures once, subculture twice, obtain corn embryonic type callus;
(4) the corn embryonic type callus obtained by step (3) is transferred on differential medium and is obtained the corn seedling of growing thickly;
(5) corn step (4) the obtained seedling of growing thickly is divided into single seedling, proceeds to regeneration whole plant in root media.
In described step (1), step (2), step (3), the inducing culture of N04 is: MS minimal medium 4 g/L(are purchased from Sigma company, article No. is m0404)+N6 vitamin (1000x) 1ml+2,4-D 0.4 mg/L+L-PROLINE 5 g/L+sucrose 30 g/L+inositol 0.1 g/L+caseinhydrolysate 0.1 g/L+AgNO
336 μ M/L+plant gel 3 g/L, pH is 5.8.
In described step (1), step (2), step (3), the inducing culture of N10 is: MS minimal medium 4 g/L(are purchased from Sigma company, article No. is m5519)+N6 vitamin (1000x) 1ml+2,4-D 2 mg/L+L-PROLINE 2.76 g/L+sucrose 30 g/L+inositol 0.1 g/L+caseinhydrolysate 0.1 g/L+AgNO
313 μ M/L+plant gel 3 g/L, pH is 5.8.
Differential medium in described step (4) is: MS minimal medium 4 g/L(are purchased from Sigma company, and article No. is m5519)+inositol 0.1 g/L+sucrose 30 g/L+6-BA 0.2 mg/L+plant gel 3 g/L, pH is 5.8.
In described step (5), root media is: MS minimal medium 4 g/L(are purchased from Sigma company, and article No. is m5519)+inositol 0.1 g/L+sucrose 30 g/L+NAA 0.2 mg/L+plant gel 30 g/L, pH is 5.8.
Inoculation and cultivation: get the pollination young fringe of latter 10-11 days, 70% alcohol carries out successively sterilization treatment to bract, peel off the immature ear of bract and put into superclean bench, clorox sterilization treatment with 5%, after 20 minutes, take out young fringe, cut the endosperm of half with scalpel, then allocation rataria, plumular axis is towards being placed down on inducing culture.27 ± 1 ℃ of dark cultivations after 3-5 days can be seen the expanded plumule that grows of rataria, cut plumule, and expanded rataria is transferred on fresh inducing culture, and 2-3 can see the embryonic type callus of formation after week.The embryonic type callus formed just can be transferred on differential medium, and differentiation is cultivated about one week, in the embryonic type callus, shows to see have green bud point to form.Continue to cultivate after one week, start to have the seedling of growing thickly to break up out, the seedling of growing thickly is separated, and each seedling is transferred to respectively the root media root induction.
In the present invention, callus induces, the differentiation of callus and culture of rootage are all carried out indoor, cultivation stage at callus, do not need the dark cultivation tune condition of illumination to be: temperature: 27 ± 1 ℃, incubation time: 24h, adopt the illumination cultivation chamber to cultivate at Calli Differentiation and culture of rootage stage, cultivation temperature: 27 ± 1 ℃, intensity of illumination: 1500-2000lx, illumination every day 14h.
Claims (5)
1. the method for pop corn rataria callus induction and plant regeneration, is characterized in that, first the corn inbred line rataria induced to callus, through twice subculture, choose wherein corn embryonic type callus, Direct Regeneration becomes whole plant, described corn inbred line is N10 or N04, and its step is as follows:
(1) after getting the immature ear sterilization treatment that the artificial autocopulation pollinates rear 10-11 days, allocation rataria in superclean bench, plumular axis, towards being placed down on inducing culture, induces maize calli for the first time;
(2) maize calli for the first time obtained by step (1) cuts plumule and transfers on new inducing culture, after 15 days, obtains maize calli for the second time;
(3) after the maize calli for the second time obtained by step (2) is caught broken, transfer on new inducing culture, every 15 days subcultures once, subculture twice, obtain corn embryonic type callus;
(4) the corn embryonic type callus obtained by step (3) is transferred on differential medium and is obtained the corn seedling of growing thickly;
(5) corn step (4) the obtained seedling of growing thickly is divided into single seedling, proceeds to regeneration whole plant in root media.
2. the method for pop corn rataria callus induction according to claim 1 and plant regeneration, it is characterized in that: in described step (1), step (2), step (3), the inducing culture of N04 is: MS minimal medium 4 g/L+N6 vitamin 1 ml+2,4-D 0.4 mg/L+L-PROLINE 5 g/L+sucrose 30 g/L+inositol 0.1 g/L+caseinhydrolysate 0.1 g/L+AgNO
336 μ M/L+plant gel 3 g/L, pH is 5.8.
3. the method for pop corn rataria callus induction according to claim 1 and plant regeneration, it is characterized in that: in described step (1), step (2), step (3), the inducing culture of N10 is: MS minimal medium 4 g/L+N6 vitamin 1ml+2,4-D 2 mg/L+L-PROLINE 2.76 g/L+sucrose 30 g/L+inositol 0.1 g/L+caseinhydrolysate 0.1 g/L+ AgNO
313 μ M/L+plant gel 3 g/L, pH is 5.8.
4. the method for pop corn rataria callus induction according to claim 1 and plant regeneration, it is characterized in that: the differential medium in described step (4) is: MS minimal medium 4 g/L+inositol 0.1 g/L+sucrose 30 g/L+6-BA 0.2 mg/L+plant gel 3 g/L, pH is 5.8.
5. the method for pop corn rataria callus induction according to claim 1 and plant regeneration, it is characterized in that: in described step (5), root media is: MS minimal medium 4 g/L+inositol 0.1 g/L+sucrose 30 g/L+NAA 0.2 mg/L+plant gel 30 g/L, pH is 5.8.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109042297A (en) * | 2018-08-21 | 2018-12-21 | 扬州大学 | A kind of corn inbred line SL1303 rataria method for transformation |
| CN111448991A (en) * | 2020-05-22 | 2020-07-28 | 上海市农业科学院 | Culture method for inducing embryogenic callus by using young waxy corn embryos |
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| DE10201637A1 (en) * | 2002-01-17 | 2003-08-07 | Univ Albert Ludwigs Freiburg | Preparing maize callus cultures, useful for regeneration of whole plants, comprises recovering coleoptile leaf tissue of seedlings and inducing callus formation by culturing in presence of proline, silver nitrate and auxins |
| CN101779598A (en) * | 2010-01-19 | 2010-07-21 | 河北省农林科学院谷子研究所 | Method for building high-efficiency regeneration system of superior corn self-bred line agriculture line 531 |
| CN102217533A (en) * | 2011-04-14 | 2011-10-19 | 中国农业大学 | Method for preparing corn type II embryogenic calli |
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2013
- 2013-09-06 CN CN201310401998.1A patent/CN103477980A/en active Pending
Patent Citations (5)
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| US20020104131A1 (en) * | 1998-12-01 | 2002-08-01 | Stine Biotechnology | Methods for tissue culturing and transforming elite inbreds of Zea mays L. |
| DE10201637A1 (en) * | 2002-01-17 | 2003-08-07 | Univ Albert Ludwigs Freiburg | Preparing maize callus cultures, useful for regeneration of whole plants, comprises recovering coleoptile leaf tissue of seedlings and inducing callus formation by culturing in presence of proline, silver nitrate and auxins |
| CN101779598A (en) * | 2010-01-19 | 2010-07-21 | 河北省农林科学院谷子研究所 | Method for building high-efficiency regeneration system of superior corn self-bred line agriculture line 531 |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109042297A (en) * | 2018-08-21 | 2018-12-21 | 扬州大学 | A kind of corn inbred line SL1303 rataria method for transformation |
| CN109042297B (en) * | 2018-08-21 | 2022-04-19 | 扬州大学 | Maize inbred line SL1303 young embryo transformation method |
| CN111448991A (en) * | 2020-05-22 | 2020-07-28 | 上海市农业科学院 | Culture method for inducing embryogenic callus by using young waxy corn embryos |
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Application publication date: 20140101 |