CN102217533B - Method for preparing corn type II embryogenic calli - Google Patents
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Abstract
The invention discloses a method for preparing corn type II embryogenic calli. With the method, a corn inbred line which can produce type II embryogenic calli can be prepared. the method comprises the flowing steps: carrying out selfing among an initial corn inbred line to obtain seeds of an F1 generation; carrying out embryogenic callus induction upon embryos of the F1 generation seeds, such that embryogenic calli are obtained; collecting type II embryogenic calli; carrying out regenerating and cultivating upon the type II embryogenic calli until corn plants are obtained. The obtained corn plants form a target corn inbred line. With type II callus phenotype, the corn material provided by the present invention is easy to regenerate, and the regenerated plants have excellent agronomic properties. According to the present invention, a novel material for present transgenic acceptors is provided, a limitation that presently only non-inbred line Hill are utilized is solved, and the period for cultivating new species of corn by using transgenic technologies is shortened.
Description
Technical field
The present invention relates to a kind of method for preparing corn II type embryo callus.
Background technology
Most of corn inbred lines can form fine and close I type callus, and this callus embryo property is poor, more difficultly behind the subculture bear plant; Crisp II type callus growth is rapid, can long-term subculture and keep embryo property.Although inducing of corn II type callus receives the genotype restriction big, because its callus ratio is easier to obtain, regeneration capacity is stronger, and the cycle that obtains transformed plant is shorter relatively, is still one of present most widely used acceptor material.
The regeneration capacity of clear and definite explant is controlled by inherent cause at present, and the difference of different genotype rataria embryo callus subculture inductivity has had a large amount of paper reports.Mostly the corn material of at present agriculture bacillus mediated genetic transformation is that A188, B73 and deriving of they are to receive genotypic restriction to a great extent.
Hodges discoveries such as (1986); A188 is the easiest to induce embryo callus and regeneration capacity also is significantly higher than other strain; With it is that parent's filial generation also shows higher somatic cell regeneration capacity; Inbred line A188 shows the most outstanding genotype, but the performance of the economical character of this strain is very poor, and the purpose proterties needs just can change in the corn variety of production value through long hybridization transformation.
Along with the day of the grain and the energy is becoming tight, the development of high oil corn will be paid close attention to.The research report of the rataria callus culture of at present relevant high oil corn self interweaving series is less.Therefore be necessary to set up a good high oil corn regenerating system, cultivate regeneration capacity strong, have high oiliness shape, material that economical character is good, for high light wood material genetic transformation lays the foundation.
I type callus: growth rate is slow, compact structure, and seedling differentiation can not be grown by long-term subculture easily, can be converted into II type callus sometimes; II type callus: fast growth, loose frangible, lovely luster, very easily seedling differentiation can be grown by long-term subculture.III type callus: growth rate is slow, short texture, and color and luster is dim, becomes water stain shape sometimes, no regeneration capacity.General in the world distinguishes I type callus and II type callus with this kind method, and the two is easy to distinguish.
Summary of the invention
An object of the present invention is to provide a kind of method for preparing the corn inbred line that can produce II type embryo callus.
Preparation provided by the present invention can produce the method for the corn inbred line of II type embryo callus, comprises the steps: the corn inbred line that sets out is carried out selfing, obtains F1 for seed; Get F1 and carry out embryonic callus induction, obtain embryo callus, picking II type embryo callus for the rataria of seed; Said II type embryo callus regenerating and culturing is obtained milpa, and the milpa that obtains is the purpose corn inbred line.
In the said process, the method for said embryonic callus induction comprises the steps: said F1 is inoculated on the embryonic callus induction medium for the rataria of seed, carries out embryonic callus induction and cultivates;
In above-mentioned arbitrary said process, said embryonic callus induction culture condition is in 28 ℃ dark surrounds, to cultivate for 2 weeks;
In above-mentioned arbitrary said process, said embryonic callus induction medium is by N6 medium, 2, and 4-D, inositol, proline, sucrose, caseinhydrolysate and plant gel are formed;
2; The concentration of 4-D in said inducing culture is 2.0-2.5mg/l; The concentration of inositol in said inducing culture is 0.1-0.17g/l; The concentration of proline in said inducing culture is 2.6-2.9g/l, and the concentration of sucrose in said inducing culture is 30g/l, and the concentration of caseinhydrolysate in said inducing culture is 0.12-0.15g/l.
In the above-mentioned embryonic callus induction medium, the concentration of plant gel in said inducing culture is 8g/l, and it is too hard to be generally the too high medium of 8g/l, is not easy to Nutrient Absorption; It is too soft to cross low medium, and explant is prone to the mobile growth that is unfavorable for.
In above-mentioned arbitrary said process, the said method that said II type embryo callus regenerating and culturing is obtained milpa comprises the steps: that said II type embryo callus is carried out embryoid induction to be cultivated, and obtains embryoid; Said embryoid is received on the differential medium, carried out differentiation culture, obtain milpa;
In above-mentioned arbitrary said process, the condition of said differentiation culture is under 28 ℃, light intensity 2000lx-2300lx, illumination every day 14h-18h condition, to cultivate 13-16 days;
In above-mentioned arbitrary said process, said differential medium is made up of MS medium, inositol, sucrose, 6-BA and plant gel;
The concentration of inositol in said differential medium is 0.1-0.17g/l, and the concentration of sucrose in said differential medium is 30g/L-35g/L, and the concentration of 6-BA in said differential medium is 0mg/L-0.5mg/L.
The concentration of plant gel in said differential medium is 8g/l, and it is too hard to be generally the too high medium of 8g/l, is not easy to Nutrient Absorption; It is too soft to cross low medium, and explant is prone to the mobile growth that is unfavorable for.
In above-mentioned arbitrary said process, said embryoid induction cultured method comprises the steps: said II type embryo callus is received on the embryoid induction medium, carries out embryoid induction and cultivates;
In above-mentioned arbitrary said process, said embryoid induction culture condition is 26-29 ℃ of dark the cultivation 14-16 days;
Said embryoid induction medium is made up of MS medium, inositol, sucrose and plant gel, and the concentration of inositol in said embryoid induction medium is 0.1-0.17g/l, and the concentration of sucrose in said embryoid induction medium is 60g/L-65g/L.
The concentration of plant gel in said embryoid induction medium is 8g/l, and it is too hard to be generally the too high medium of 8g/l, is not easy to Nutrient Absorption; It is too soft to cross low medium, and explant is prone to the mobile growth that is unfavorable for.
In above-mentioned arbitrary said process, the said corn inbred line that sets out is high oily inbred line GY302.
Another object of the present invention provides a kind of method for preparing the II type embryo callus of corn.
The method of the II type embryo callus of preparation corn provided by the present invention comprises the steps:
The corn inbred line method that 1) can produce II type embryo callus according to above-mentioned arbitrary preparation prepares the corn inbred line that can produce II type embryo callus;
2) get the said rataria that can produce the corn inbred line of II type embryo callus, carry out embryonic callus induction, obtain the II type embryo callus of corn.
In the process of the II type embryo callus of above-mentioned preparation corn, the method for said embryonic callus induction comprises the steps: said rataria is inoculated on the embryonic callus induction medium, carries out embryonic callus induction and cultivates;
In the process of the II type embryo callus of above-mentioned preparation corn, said embryonic callus induction culture condition is in 28 ℃ dark surrounds, to cultivate for 2 weeks;
In the process of the II type embryo callus of above-mentioned preparation corn, said embryonic callus induction medium is by N6 medium, 2, and 4-D, inositol, proline, sucrose, caseinhydrolysate and plant gel are formed;
2; The concentration of 4-D in said inducing culture is 2.0-2.5mg/l; The concentration of inositol in said inducing culture is 0.1-0.17g/l; The concentration of proline in said inducing culture is 2.6-2.9g/l, and the concentration of sucrose in said inducing culture is 30g/l, and the concentration of caseinhydrolysate in said inducing culture is 0.12-0.15g/l;
The concentration of plant gel in said inducing culture is 8g/l.It is too hard to be generally the too high medium of 8g/l, is not easy to Nutrient Absorption; It is too soft to cross low medium, and explant is prone to the mobile growth that is unfavorable for.
The inventive method has obtained having II type callus phenotype, easy, the good corn material of regeneration plant economical character of regeneration; For present genetically modified acceptor provides new material; Break the limitation that only limits to utilize a few inbred lines such as Hill, AB inbred line at present, and shortened the time of utilizing transgenic technology to breed new varieties of corn.
Description of drawings
Fig. 1 is the tassel proterties of NGY302.
Fig. 2 is the tree characteristics of NGY302.
Embodiment
Employed experimental technique is conventional method like no specified otherwise among the following embodiment.
Used material, reagent etc. like no specified otherwise, all can obtain from commercial sources among the following embodiment.
High oily inbred line GY302 document " Zhang Yang, Jiang Haiying, Liu Ming, Zhang Baoshi. the coordinate force of high oil corn self interweaving series yield traits and cluster analysis. corn science .2006,14 (3) " in disclosed, the public can obtain from China Agricultural University.
Embodiment 1, the preparation and the checking that can produce the corn inbred line-NGY302 of II type embryo callus
One, preparation and checking
(1) preparation
1, the oily inbred line GY302 of height is carried out selfing, obtain F1 for seed;
In mid-August, 2009 is the experiment of China Agricultural University experiment station in Changping, Beijing, and oily inbred line GY302 carries out individual plant selfing with height, obtains selfing F1 for seed.
2, II type embryonic callus induction:
Get 100 F1 and carry out embryonic callus induction, obtain 100 embryo callus, picking II type embryo callus therefrom, the inductivity of statistics II type embryo callus for the rataria of seed.
The embryonic callus induction method: with 10 days corncob behind the pollination self of high light wood material GY302 field with 10% aqueous sodium hypochlorite solution sterilization 10min; Clean 2 times with aqua sterilisa again; In superclean bench, rataria is stripped out; In being inoculated on the superclean bench on the embryonic callus induction medium, be positioned over cultivated for 2 weeks in 28 ℃ the dark surrounds after, obtain embryo callus.
Said embryo callus medium is by N6 medium, 2, and 4-D, inositol, proline, sucrose, caseinhydrolysate and plant gel are formed; 2; The concentration of 4-D in inducing culture is 2.2mg/l; The concentration of inositol in inducing culture is 0.15g/l, and the concentration of proline in inducing culture is 2.8g/l, and the concentration of sucrose in inducing culture is 30g/l; The concentration of caseinhydrolysate in inducing culture is 0.15g/l, and the concentration of plant gel in inducing culture is 8g/l.The N6 medium is available from Sigma, and catalog number is C1416.
The method of discrimination of II type callus: fast growth, loose frangible, lovely luster, very easily seedling differentiation can be grown by long-term subculture.Other between between I type and the II type or the callus between II type and III type all do not remember the II type of doing.
The callus subculture method: subculture medium is identical with above-mentioned embryonic callus induction medium.Successive transfer culture condition: in 28 ℃ dark surrounds, cultivated for 2 weeks.
The result: the II type embryo callus fast growth of choosing, organize loosely, rough, be graininess, lovely luster, subculture 10 times, still be II type embryo callus; Choose 2 II type embryo callus; The inductivity of II type embryo callus is 2%.
3, regeneration:
Said II type embryo callus regenerating and culturing is obtained milpa, and the milpa that obtains is the purpose corn inbred line, and note is made NGY302.The statistics regeneration rate.
Successive transfer culture for the first time: the initial II type embryo callus successive transfer culture that step 2 is obtained; The medium of successive transfer culture is with consistent described in successive transfer culture condition and the step 2; The callus that obtains behind the successive transfer culture still is II type embryo callus, and note is made subculture II type embryo callus for the first time;
Successive transfer culture for the second time: subculture II type embryo callus morsel for the first time, every is carried out successive transfer culture again, and the medium of successive transfer culture obtains 150 II type embryo callus with consistent described in successive transfer culture condition and the step 2.
Embryoid induction: 150 II type callus are received on the embryoid induction medium, and 28 ℃ of dark cultivations 15 days obtain white embryoid;
Said embryoid induction medium is made up of MS medium, inositol, sucrose and plant gel; The concentration of inositol in said embryoid induction medium is 0.15g/l; The concentration of sucrose in said embryoid induction medium is 63g/L, and the concentration of plant gel in said embryoid induction medium is 8g/l;
Plant differentiation: white embryoid is received on the differential medium, under 28 ℃, light intensity 2200lx, illumination every day 16h condition, cultivated 14 days; Obtain the regeneration plant seedling.
Said differential medium is made up of MS medium, inositol, sucrose, 6-BA and plant gel; The concentration of inositol in said differential medium is 0.15g/l, and the concentration of sucrose in said differential medium is 33g/L, and the concentration of 6-BA in said differential medium is 0.3mg/L, and the concentration of plant gel in said differential medium is 8g/l;
The regeneration plant seedling is moved to big Tanaka, and growth obtains ripe milpa, and note is made NGY302.
Inducing embryoid body rate: total II type callus number=130 white embryoid/150 II type callus=86.67% that obtain the callus number of white embryoid/be used to induce.
Embryoid regeneration rate: obtain the white embryoid number of plant seedling/be used for total white embryoid number=85 plant/130 embryoid=65.38% of differentiation culture.
(2) checking of NGY302
1, produces the stability of II type embryo callus
Checking 1: in March, 2010, in the China Agricultural University greenhouse, NGY302 is carried out selfing, obtain F1 for seed; According to the method for experiment in one in July, 2010; Experiment station, the village in China Agricultural University; Get F1 and carry out embryonic callus induction, obtain embryo callus (note is made F1 for embryo callus), statistics II type frequency of embryonic callus induction for selfing gained seed rataria; II type frequency of embryonic callus induction is 100% as a result.
Checking 2: in November, 2010, the China Agricultural University experiment station in Hainan verifies that according to method described in the checking 1 II type frequency of embryonic callus induction is 100% as a result.
Comprehensive above checking result shows that NGY302 produces the stable very good of II type embryo callus.This meets the principle of the phenotype of generation II type embryo callus by Gene Handling.
2, the economical character of NGY302 detects
Test as follows in time in summer in 2010, place, the Bei Jingshang village.
NGY302 is carried out selfing, obtain F1 for seed; F1 is planted for seed, obtain newborn plant.Add up emergence rate, observe the economical character (male and female florescence, loose powder property, seed pollination ripening rate, plant plant height, blade shape isophenous) of plant, detect the oil content of fruit ear seed in the newborn plant.
According to method described in the document " Science 16 May 1969:Vol.164 no.3881 pp.827-828.DOI:10.1126/science.164.3881.827.Nuclear Magnetic Resonance Measurement of Oil " Unsaturation " in Single Viable Corn Kernels.T.F.Conway and L.F.Johnson ", utilize near-infrared analyzer to measure the oil content of embryo of the seed of NGY302.
The result:
The seed emergence rate reaches 100%.
The newborn plant male and female florescence is consistent, and loose powder property is good (at dry hot weather, shakes male flower; Flower pesticide descends slowly and lightly easily, is the dispersed particles shape, and the loose powder amount is big); Seed pollination ripening rate high (seed in the corncob of newborn plant almost 100% be full); Plant plant height medium (1.7m-1.9m), plant is strong, and blade is narrow leaf more crisp hard upright (Fig. 1,2) slightly.
Seed oil content result such as table 1.It not quite still is high oil content that the oil content of seed embryo changes.
Table 1, oil content, two kinds of 8 of material random chooses are measured oil content:
NGY302 seed oil content is measured the result
GY302 seed oil content is measured the result
3 repetitions are established in experiment, all obtain the stable NGY302 of proterties.
Two, preparation and checking
Basic identical in method and the experiment one, difference is following:
1, in the II type embryonic callus induction step, the medium of use is following:
Inducing culture: except that the following concentration difference of each material in inducing culture, all the other are all identical with the inducing culture of testing in: 2, and 4-D 2.0mg/l; Inositol 0.1g/l, proline 2.6g/l, sucrose 30g/l; Caseinhydrolysate 0.12g/l, plant gel 8g/l.
2, in the embryoid induction step,
The medium that uses is following: said embryoid induction medium is made up of MS medium, inositol, sucrose and plant gel; The concentration of inositol in said embryoid induction medium is 0.1g/l; The concentration of sucrose in said embryoid induction medium is 60g/L, and the concentration of plant gel in said embryoid induction medium is 8g/l;
Said embryoid induction culture condition is 26 ℃ of dark cultivations 14 days;
3, in the plant differentiation step,
The medium that uses is following: except that the following concentration difference of each material in differential medium; All the other are all identical with the differential medium of testing in: the concentration of inositol in said differential medium is 0.1g/l; The concentration of sucrose in said differential medium is 30g/L; The concentration of 6-BA in said differential medium is 0mg/L, and the concentration of plant gel in said differential medium is 8g/l;
The condition of said differentiation culture is under 28 ℃, light intensity 2000lx, illumination every day 14h condition, to cultivate 13 days;
3 repetitions are established in experiment, and the result obtains identical corn inbred line with experiment one.
Three, preparation and checking
Basic identical in method and the experiment one, difference is following:
1, in the II type embryonic callus induction step, the medium of use is following:
Inducing culture: except that the following concentration difference of each material in inducing culture, all the other are all identical with the inducing culture of testing in: 2, and 4-D 2.5mg/l; Inositol 0.17g/l, proline 2.9g/l, sucrose 30g/l; Caseinhydrolysate 0.14g/l, plant gel 8g/l.
2, in the embryoid induction step,
The medium that uses is following: said embryoid induction medium is made up of MS medium, inositol, sucrose and plant gel; The concentration of inositol in said embryoid induction medium is 0.17g/l; The concentration of sucrose in said embryoid induction medium is 65g/L, and the concentration of plant gel in said embryoid induction medium is 8g/l;
Said embryoid induction culture condition is 29 ℃ of dark cultivations 16 days;
3, in the plant differentiation step,
The medium that uses is following: except that the following concentration difference of each material in differential medium; All the other are all identical with the differential medium of testing in: the concentration of inositol in said differential medium is 0.17g/l; The concentration of sucrose in said differential medium is 35g/L; The concentration of 6-BA in said differential medium is 0.5mg/L, and the concentration of plant gel in said differential medium is 8g/l;
The condition of said differentiation culture is under 28 ℃, light intensity 2300lx, illumination every day 18h condition, to cultivate 16 days;
3 repetitions are established in experiment, and the result obtains identical corn inbred line with experiment one.
Embodiment 2, prepare II type embryo callus with NGY302
One, method I
The rataria of NGY302 is inoculated on the embryo callus medium, in 28 ℃ dark surrounds, cultivated for 2 weeks, obtain II type embryo callus;
Said embryo callus medium is by N6 medium, 2, and 4-D, inositol, proline, sucrose, caseinhydrolysate and plant gel are formed;
2; The concentration of 4-D in said inducing culture is 2.2mg/l; The concentration of inositol in said inducing culture is 0.15g/l, and the concentration of proline in said inducing culture is 2.8g/l, and the concentration of sucrose in said inducing culture is 30g/l; The concentration of caseinhydrolysate in said inducing culture is 0.15g/l, and the concentration of plant gel in said inducing culture is 8g/l.
The N6 medium is available from Sigma, and catalog number is C1416.
The rataria of cultivation that the result is useful on all obtains II type embryo callus.
Two, method II
The rataria of NGY302 is inoculated on the embryo callus medium, in 28 ℃ dark surrounds, cultivated for 2 weeks, obtain II type embryo callus;
Said embryo callus medium is by N6 medium, 2, and 4-D, inositol, proline, sucrose, caseinhydrolysate and plant gel are formed;
2; The concentration of 4-D in said inducing culture is 2.0mg/l; The concentration of inositol in said inducing culture is 0.1g/l, and the concentration of proline in said inducing culture is 2.6g/l, and the concentration of sucrose in said inducing culture is 30g/l; The concentration of caseinhydrolysate in said inducing culture is 0.12g/l, and the concentration of plant gel in said inducing culture is 8g/l.
The N6 medium is available from Sigma, and catalog number is C1416.
The rataria of cultivation that the result is useful on all obtains II type embryo callus.
Three, method III
The rataria of NGY302 is inoculated on the embryo callus medium, in 28 ℃ dark surrounds, cultivated for 2 weeks, obtain II type embryo callus;
Said embryo callus medium is by N6 medium, 2, and 4-D, inositol, proline, sucrose, caseinhydrolysate and plant gel are formed;
2; The concentration of 4-D in said inducing culture is 2.5mg/l; The concentration of inositol in said inducing culture is 0.17g/l, and the concentration of proline in said inducing culture is 2.9g/l, and the concentration of sucrose in said inducing culture is 30g/l; The concentration of caseinhydrolysate in said inducing culture is 0.14g/l, and the concentration of plant gel in said inducing culture is 8g/l.
The N6 medium is available from Sigma, and catalog number is C1416.
The rataria of cultivation that the result is useful on all obtains II type embryo callus.
Embodiment 3, contrast
A188 disclosed in document " corn Somatic Cell Culture and the screening of disease-resistant variant, 1993 ", and the public can obtain from China Agricultural University.
Summer in 2010, the seed of A188 and the seed of NGY302 are planted the experiment centre in the Bei Jingshang village respectively, observe its economical character.The A188 plant is short and small as a result, and the pollination ripening rate is about 80%, and the performance of male and female florescence is inharmonious, is separated by 2 days; And the NGY302 plant is strong, and is highly medium, and the pollination ripening rate is high, is about 100%.
The seed rataria of A188 and the seed rataria of NGY302 are planted respectively on the N6 medium, be positioned in 28 ℃ the dark surrounds and cultivated for 2 weeks, observable callus still is the II type for the I type.
A188 is in same N6 cultivation, and the callus performance 95% of inducing is between I, II; NGY302 then is a 100%II type callus.
In being divided into the incubation step of plant, A188 need add the 6-BA somatotropin of certain concentration on the MS medium, just can differentiate the plant seedling; NGY302 then need not to add 6-BA and gets final product.
Claims (6)
1. a method for preparing the corn inbred line that can produce II type embryo callus comprises the steps: the corn inbred line that sets out is carried out selfing, obtains F1 for seed; Get F1 and carry out embryonic callus induction, obtain embryo callus, picking II type embryo callus for the rataria of seed; Said II type embryo callus regenerating and culturing is obtained milpa, and the milpa that obtains is the purpose corn inbred line;
The said method that said II type embryo callus regenerating and culturing is obtained milpa comprises the steps: said II type embryo callus is received on the embryoid induction medium, carries out embryoid induction and cultivates, and obtains embryoid; Said embryoid is received on the differential medium, carried out differentiation culture, obtain milpa;
The said corn inbred line that sets out is high oily inbred line GY302;
Said embryonic callus induction medium is by N6 medium, 2, and 4-D, inositol, proline, sucrose, caseinhydrolysate and plant gel are formed; 2; The concentration of 4-D in said inducing culture is 2.0-2.5mg/L; The concentration of inositol in said inducing culture is 0.1-0.17g/L; The concentration of proline in said inducing culture is 2.6-2.9g/L, and the concentration of sucrose in said inducing culture is 30g/L, and the concentration of caseinhydrolysate in said inducing culture is 0.12-0.15g/L;
Said embryoid induction medium is made up of MS medium, inositol, sucrose and plant gel, and the concentration of inositol in said embryoid induction medium is 0.1-0.17g/L, and the concentration of sucrose in said embryoid induction medium is 60g/L-65g/L;
Said differential medium is made up of MS medium, inositol, sucrose, 6-BA and plant gel;
The concentration of inositol in said differential medium is 0.1-0.17g/L, and the concentration of sucrose in said differential medium is 30g/L-35g/L, and the concentration of 6-BA in said differential medium is 0mg/L-0.5mg/L.
2. method according to claim 1 is characterized in that: the method for said embryonic callus induction comprises the steps: said F1 is inoculated on the embryonic callus induction medium for the rataria of seed, carries out embryonic callus induction and cultivates;
Said embryonic callus induction culture condition is in 28 ℃ dark surrounds, to cultivate for 2 weeks.
3. method according to claim 1 and 2 is characterized in that: the condition of said differentiation culture is for cultivating 13-16 days under 28 ℃, light intensity 2000lx-2300lx, illumination every day 14h-18h condition.
4. method according to claim 1 and 2 is characterized in that: said embryoid induction culture condition is 26-29 ℃ of dark the cultivation 14-16 days.
5. a method for preparing the II type embryo callus of corn comprises the steps:
1) prepares the corn inbred line that can produce II type embryo callus according to arbitrary said method among the claim 1-4;
2) get the said rataria that can produce the corn inbred line of II type embryo callus, carry out embryonic callus induction, obtain the II type embryo callus of corn.
6. method according to claim 5 is characterized in that: the method for said embryonic callus induction comprises the steps: said rataria is inoculated on the embryonic callus induction medium, carries out embryonic callus induction and cultivates;
Said embryonic callus induction culture condition is in 28 ℃ dark surrounds, to cultivate for 2 weeks;
Said embryonic callus induction medium is by N6 medium, 2, and 4-D, inositol, proline, sucrose, caseinhydrolysate and plant gel are formed;
2; The concentration of 4-D in said inducing culture is 2.0-2.5mg/L; The concentration of inositol in said inducing culture is 0.1-0.17g/L; The concentration of proline in said inducing culture is 2.6-2.9g/L, and the concentration of sucrose in said inducing culture is 30g/L, and the concentration of caseinhydrolysate in said inducing culture is 0.12-0.15g/L.
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| CN109042297B (en) * | 2018-08-21 | 2022-04-19 | 扬州大学 | Maize inbred line SL1303 young embryo transformation method |
| CN111154797B (en) * | 2020-01-22 | 2022-03-01 | 北京市农林科学院 | Genetic transformation method of maize backbone inbred line mediated by gene gun |
| CN111616047B (en) * | 2020-05-21 | 2022-06-21 | 中国农业大学 | Method for doubling corn haploid and application thereof |
| CN111448991B (en) * | 2020-05-22 | 2022-08-05 | 上海市农业科学院 | Culture method for inducing embryogenic callus by using young waxy corn embryos |
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| CN101011031A (en) * | 2007-02-07 | 2007-08-08 | 华中农业大学 | Callus induction and plant regeneration method by corn mature embryo approach |
| 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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| CN101011031A (en) * | 2007-02-07 | 2007-08-08 | 华中农业大学 | Callus induction and plant regeneration method by corn mature embryo approach |
| 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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