CN101960988A - Method for inducing adventitious buds of peanuts - Google Patents
Method for inducing adventitious buds of peanuts Download PDFInfo
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- CN101960988A CN101960988A CN 201010298256 CN201010298256A CN101960988A CN 101960988 A CN101960988 A CN 101960988A CN 201010298256 CN201010298256 CN 201010298256 CN 201010298256 A CN201010298256 A CN 201010298256A CN 101960988 A CN101960988 A CN 101960988A
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Abstract
The invention relates to a method for inducing adventitious buds of peanuts highly effectively. The method comprises the following steps of: selecting mature Yuhua No.1 peanut seeds; infiltrating the peanut seeds with 70 percent ethanol first; and then, sterilizing the peanut seeds with 0.1 percent HgCl2 solution; washing the peanut seeds with sterile water; soaking the peanut seeds in the sterile water; inoculating the other halves of remaining cotyledons with plumules into a hormone-free MS culture medium for culturing; culturing for 4 days; transversely cutting the cultured spires; dividing each spire into three parts uniformly to obtain explants; inducing and differentiating, wherein green buds appear on the edges of the explants after inoculating the explants into the culture medium for culturing 7 to 8 days; transferring the explants into a subculture medium; and culturing for 18 to 22 days again to obtain induced buds. In the method, the adventitious buds of the peanuts are obtained by selecting genotype Yuhua No.1 as the explants, which optimizes a formula of a bud induction medium; and a plurality of inoculation experiments show that the bud induction rate is stabilized over 90 percent and the repeatability is high.
Description
Technical field
The present invention relates to a kind of tissue culture and induce the grow thickly method of bud of peanut, particularly relate to a kind of method of efficiently inducing the peanut indefinite bud, belong to biological technical field.
Background technology:
Peanut is a kind of important oil crop and economic crops, and cultivated area is bigger in China.Improve output, the quality of peanut, be unable to do without improved seeds, the peanut tissue culture is to utilize biotechnology to carry out an important step of peanut breeding.
At present, some crop has been set up plant regeneration system efficiently, filter out high inductivity genotype (as paddy rice Japanese fine, China 11), but the shoot regeneration frequency of most of crops still very low (1%-30%) becomes the bottleneck that the restriction gene engineering is used.What peanut inducing clumping bud rate was reported at present on average is no more than 50%, and stability and poor repeatability.The foundation of high-efficiency peanut regenerating system is the basis of peanut genetic transformation.It is very big that plant regeneration is influenced by genotype, simultaneously also relevant with explant type and culture medium prescription.Relatively effectively, BA and TDZ are at multiple work in the regeneration of tissue culture induced bud for the basic element of cell division
Summary of the invention:
The technical problem to be solved in the present invention: provide a kind of inductivity height, stability tissue culture high, good reproducibility to induce the method for peanut indefinite bud.
Technical scheme of the present invention:
A kind of method of inducing the peanut indefinite bud comprises the steps:
(1) seed pre-culture is chosen ripe Henan and is spent peanut seed No. 1, soaks into 20-30s with 70% ethanol earlier, uses 0.1% HgCl then
2Solution disinfection 8-10min, with aseptic water washing 5 ~ 6 times, in sterile water, soak 2 ~ 3h then, peanut kind skin is removed, remove a slice cotyledon again, remaining second half cotyledon that has plumule is inoculated in the MS medium that does not add hormone cultivates, 25 ~ 26 ℃ of cultivation temperature, light application time 12-14h/d, intensity of illumination 1000-2000 lx cultivated 4 days;
(2) the peanut cotyledon after the explant preparation will be cultivated gets rid of, and clamps plumular axis, with sterile razor blade the peanut spire is downcut from base portion, with the spire crosscut, is divided into three parts, or is divided into two from nearly petiole 1/3, obtains explant;
(3) induce and break up explant is seeded in the inducing culture and cultivate, cultivation temperature is 25 ~ 26 ℃, light application time 12-14h/d, intensity of illumination 1000-2000 lx, cultivate that green bud point appears in the explant edge after 7-8 days, forward in the subculture medium, cultivate again and obtained induced bud in 18-22 days.
Described inducing culture is: MS+BA 3 mg/L+NAA 0.4-0.8 mg/L+TDZ 0.1-0.3 mg/L+AgNO
33.5 mg/L.
Described subculture medium is MS+BA 5 mg/L+NAA 1mg/L+AgNO
33.5 mg/L.
Beneficial effect of the present invention:
(1) the present invention is by strict screening, select genotype Henan and spend No. 1 as explant, through the preparation of seed pre-culture, explant and induce, link such as differentiation, obtain the peanut indefinite bud, than the grow thickly inductivity height of bud of the peanut of present bibliographical information, prove that through inoculation test repeatedly the inductivity result is stable, repeatability better.
(2) the present invention has optimized bud inducing culture based formulas, and the time that makes explant sprout indefinite bud shifts to an earlier date greatly, and sprout time advanceed to 7-8 days from original about 14 days.
(3) the inventive method can obtain stable high inductivity, and cultivation (MS+BA 3 mg/L+NAA 0.4 mg/L+TDZ 0.3 mg/L+AgNO on three kinds of different medium are spent in Henan No. 1
33.5 mg/L; MS+BA 3 mg/L+NAA 0.8 mg/L+TDZ 0.1 mg/L+AgNO
33.5 mg/L; MS+BA 3 mg/L+NAA0.8 mg/L+TDZ 0.3 mg/L+AgNO
33.5 mg/L), bud induction rate is stabilized in more than 90%, is respectively 91.6 ~ 92.5%, 97.5 ~ 98.5%, 99.5 ~ 100%.
And open farming 49 under the similarity condition, open farming 30, Henan spends No. 15 at MS+BA 3 mg/L+NAA0.8 mg/L+TDZ 0.3 mg/L+AgNO
33.5 cultivate on the mg/L medium, its adventitious bud induction frequency is respectively 51.2 ~ 52.6%, 31.8 ~ 33.5%, 36.8 ~ 38.5%.Can find out that Henan spends No. 1 adventitious bud induction frequency during as explant more obvious than other species the advantage.
Description of drawings:
Fig. 1: Henan spends No. 1 explant at MS+BA 3 mg/L+NAA0.8 mg/L+TDZ 0.3 mg/L+AgNO
33.5 evoking adventive bud situation on the mg/L medium.The green projection of explant notching edge is the indefinite bud of inducing generation among the figure.
Fig. 2: open agricultural 49(contrast) explant is at MS+BA 3 mg/L+NAA0.8 mg/L+TDZ 0.3 mg/L+AgNO
33.5 evoking adventive bud situation on the mg/L medium.The green projection of explant notching edge is the indefinite bud of inducing generation among the figure, and yellow, white is callus.
From Fig. 1,2 as can be seen, under identical medium and condition of culture, Henan spends the bud point of No. 1 explant many and growing way is prosperous; Open agricultural 49(contrast) explant bud point is rare, and the explant edge produces white loose callus, illustrates that spend No. 1 in Henan and there is significant difference in the bud induction rate opened between agricultural 49 genotype.
Fig. 3: No. 15 (contrast) explant induction indefinite bud contrast figure on three kinds of different medium is spent in Henan: the green projection of explant notching edge is the indefinite bud of inducing generation among the figure, and yellow, white is callus.
(1) medium is MS+BA 3 mg/L+NAA0.8 mg/L+TDZ 0.3 mg/L+AgNO among the figure
33.5 mg/L; (2) medium is MS+BA 3 mg/L+NAA 0.8 mg/L+TDZ 0.1 mg/L+AgNO
33.5 mg/ L; (3) medium is MS+BA 3 mg/L+NAA 0.4 mg/L+TDZ 0.3 mg/L+AgNO
33.5 mg/L.
As can be seen from Figure 3, same genotype (spend No. 15 in Henan) on different medium, the induced reaction difference of indefinite bud.(2) and (3) induce more callus than (1) among the figure, but adventitious bud induction frequency is all lower, inductivity less than 40%.
Embodiment:
Describe the present invention in detail below in conjunction with embodiment, wherein the percent concentration of Chu Xianing all refers to mass percent concentration if no special instructions.
Embodiment 1:Induce the method for peanut indefinite bud, comprise the steps:
1, seed pre-culture: choose full grains, mature seed is spent in big, Henan flawless, that do not go out plumule, surface No. 1, adds in the aseptic triangular flask with tweezers, with 70% ethanol infiltration 30s, uses 0.1% HgCl more earlier
2Solution disinfection 8min uses aseptic water washing 5 ~ 6 times, soaks 2 ~ 3h then in sterile water, after treating that peanut kind skin is unfolded, on superclean bench, peanut kind skin is removed, remove a slice cotyledon again, remaining second half cotyledon that has plumule is inoculated in MS is housed with tweezers
0In the aseptic Cans of (the MS medium does not add any hormone) medium, putting in the illumination cultivation chamber, is 2000lx at 25 ℃, intensity of illumination, cultivates 4 days under the illumination 14h/d condition;
2, preparation explant: get rid of with the cotyledon of tweezers, clamp plumular axis afterwards, with sterile razor blade the peanut spire is downcut from base portion again, the nearly petiole of spire portion 1/3 place is divided into two with peanut;
3, induce and break up: explant is seeded in respectively in the inducing culture, and the bud inducing culture is: MS+BA 3 mg/L+NAA 0.8 mg/L+TDZ 0.3 mg/L+AgNO
33.5 mg/L puts 25 ℃ of illumination cultivation chambers, intensity of illumination is 2000lx, cultivates under the illumination 14h/d condition, and through 7-8 days, the appearance of part explant notching edge visible green bud point; About 14 days, a large amount of green bud points all appear in all explant edges.
After two weeks, forward in the subculture medium, make the further differentiation of bud point, subculture medium is MS+BA 5 mg/L+NAA 1mg/L+AgNO
33.5 mg/L cultivates and obtained inducing the peanut indefinite bud in 18 days.By statistics, inductivity reaches 100%.
Embodiment 2:Induce the method for peanut indefinite bud
Seed pre-culture: choose full grains, mature seed is spent in big, Henan flawless, that do not go out plumule, surface No. 1, adds in the aseptic triangular flask with tweezers, with 70% ethanol infiltration 20s, uses 0.1%HgCl more earlier
2Solution disinfection 10min uses aseptic water washing 5 ~ 6 times, soaks 2 ~ 3h then in sterile water, after treating that peanut kind skin is unfolded, on superclean bench, peanut kind skin is removed, remove a slice cotyledon again, remaining second half cotyledon that has plumule is inoculated in MS is housed with tweezers
0In the aseptic Cans of (the MS medium does not add any hormone) medium.Putting in the illumination cultivation chamber, is 1500 lx at 26 ℃, intensity of illumination, cultivates 4 days under the illumination 12h/d condition.
Preparation explant: get rid of with the cotyledon of tweezers, clamp plumular axis afterwards, with sterile razor blade the peanut spire is downcut from base portion again, spire crosscut average is divided into three with peanut.
Induce and break up: explant is seeded in MS+BA 3 mg/L on the inducing culture+NAA 0.8 mg/L+TDZ 0.1 mg/L+AgNO respectively
33.5 mg/L puts 26 ℃ of illumination cultivation chambers, intensity of illumination is 1500 lx, cultivates under the illumination 12h/d condition, and through 7-8 days, the appearance of part explant notching edge visible green bud point; About 14 days, almost a large amount of green bud points all appear in the explant edge.After two weeks, forward on the subculture medium, subculture medium is MS+BA 5 mg/L+NAA 1mg/L+AgNO
33.5 mg/L makes the further differentiation of bud point, cultivates and obtains inducing the peanut indefinite bud in 22 days.By statistics, inductivity is 97.5%.
Embodiment 3:Basic identical with embodiment 1, difference is:
Culturing room's condition: 26 ℃, intensity of illumination 1000 lx, illumination 13h/d;
Bud inducing culture: MS+BA 3 mg/L+NAA 0.4 mg/L+TDZ 0.3 mg/L+AgNO
33.5 mg/L, inductivity are 91.6%.
Specification digest
The present invention relates to a kind of method of efficiently inducing the peanut adventitious bud, comprise that choosing ripe Henan spends peanut seed No. 1, infiltrate with 70% ethanol earlier, use then 0.1% HgCl2Solution disinfection is used aseptic water washing, soaks in sterilized water then, remaining second half cotyledon with plumule is inoculated in the MS culture medium that does not add hormone cultivates, and cultivates 4 days; Spire crosscut with after cultivating is divided into three parts, obtains explant; Induction and differentiation is seeded in explant in the inducing culture and cultivates that green bud point appears in the explant edge after 7-8 days, forwards in the subculture medium, cultivates and obtains induced bud in 18-22 days. The present invention selects genotype Henan and spends and obtain the peanut adventitious bud as explant No. 1, has optimized bud inducing culture prescription, proves that through inoculation test repeatedly bud induction rate is stabilized in more than 90%, good reproducibility.
Claims (3)
1. method of inducing the peanut indefinite bud, it is characterized in that: this method comprises the steps:
(1) seed pre-culture is chosen ripe Henan and is spent peanut seed No. 1, soaks into 20-30s with 70% ethanol earlier, uses 0.1% HgCl then
2Solution disinfection 8-10min, with aseptic water washing 5 ~ 6 times, in sterile water, soak 2 ~ 3h then, peanut kind skin is removed, remove a slice cotyledon again, remaining second half cotyledon that has plumule is inoculated in the MS medium that does not add hormone cultivates, 25 ~ 26 ℃ of cultivation temperature, light application time 12-14h/d, intensity of illumination 1000-2000 lx cultivated 4 days;
(2) the peanut cotyledon after the explant preparation will be cultivated gets rid of, and clamps plumular axis, with sterile razor blade the peanut spire is downcut from base portion, with the spire crosscut, is divided into three parts, or is divided into two from nearly petiole 1/3, obtains explant;
(3) induce and break up explant is seeded in the inducing culture and cultivate, cultivation temperature is 25 ~ 26 ℃, light application time 12-14h/d, intensity of illumination 1000-2000 lx, cultivate that green bud point appears in the explant edge after 7-8 days, forward in the subculture medium, cultivate again and obtained induced bud in 18-22 days.
2. method of inducing the peanut indefinite bud according to claim 1 is characterized in that: described inducing culture is: MS+BA 3 mg/L+NAA 0.4-0.8 mg/L+TDZ 0.1-0.3 mg/L+AgNO
33.5 mg/L.
3. method of inducing the peanut indefinite bud according to claim 1 and 2 is characterized in that: described subculture medium is MS+BA 5 mg/L+NAA 1mg/L+AgNO
33.5 mg/L.
?
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| CN101960988B CN101960988B (en) | 2012-07-04 |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102239807A (en) * | 2011-06-29 | 2011-11-16 | 中国农业科学院植物保护研究所 | Establishing method of peanut regenerating system by taking deembryonated cotyledon as explant |
| CN102257965A (en) * | 2011-06-29 | 2011-11-30 | 中国农业科学院植物保护研究所 | Method for establishing peanut regeneration system with young leaf as explant |
| CN102960237A (en) * | 2012-11-22 | 2013-03-13 | 河南省农业科学院 | Method for obtaining, breeding and storing peanut interspecies hybridization variety, and identifying molecular cytology |
| CN110024694A (en) * | 2019-06-04 | 2019-07-19 | 临沂大学 | A kind of method that rapid induction Peanut Leaflet is differentiated to form adventitious bud again |
| CN110367124A (en) * | 2019-08-29 | 2019-10-25 | 淮北师范大学 | A method of building peanut cotylcdon regenerating system |
| CN110809935A (en) * | 2019-11-08 | 2020-02-21 | 广东省农业科学院果树研究所 | Method for improving germination rate of banana seeds |
| CN113040052A (en) * | 2021-04-22 | 2021-06-29 | 大连民族大学 | Method for quickly constructing peanut regeneration system by taking young leaves as explants |
-
2010
- 2010-09-30 CN CN 201010298256 patent/CN101960988B/en not_active Expired - Fee Related
Non-Patent Citations (3)
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| 《华南农业大学学报(自然科学版)》 20030731 何红卫，等 花生上胚轴的丛生芽诱导和植株再生 1-3 第24卷, 第3期 2 * |
| 《植物学通报》 20030630 林荣双，等 花生幼叶为外植体的植株再生系统的建立 1-3 第20卷, 第3期 2 * |
| 《河南农业科学》 20080131 苗利娟，等 花生幼叶丛生芽高效诱导的制约因素研究 40-44 1-3 , 第1期 2 * |
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102239807A (en) * | 2011-06-29 | 2011-11-16 | 中国农业科学院植物保护研究所 | Establishing method of peanut regenerating system by taking deembryonated cotyledon as explant |
| CN102257965A (en) * | 2011-06-29 | 2011-11-30 | 中国农业科学院植物保护研究所 | Method for establishing peanut regeneration system with young leaf as explant |
| CN102239807B (en) * | 2011-06-29 | 2013-01-02 | 中国农业科学院植物保护研究所 | Establishing method of peanut regenerating system by taking deembryonated cotyledon as explant |
| CN102960237A (en) * | 2012-11-22 | 2013-03-13 | 河南省农业科学院 | Method for obtaining, breeding and storing peanut interspecies hybridization variety, and identifying molecular cytology |
| CN102960237B (en) * | 2012-11-22 | 2013-12-18 | 河南省农业科学院 | Method for obtaining, breeding and storing peanut interspecies hybridization variety, and identifying molecular cytology |
| CN110024694A (en) * | 2019-06-04 | 2019-07-19 | 临沂大学 | A kind of method that rapid induction Peanut Leaflet is differentiated to form adventitious bud again |
| CN110024694B (en) * | 2019-06-04 | 2022-02-15 | 临沂大学 | Method for rapidly inducing redifferentiation of peanut young leaves to form adventitious buds |
| CN110367124A (en) * | 2019-08-29 | 2019-10-25 | 淮北师范大学 | A method of building peanut cotylcdon regenerating system |
| CN110367124B (en) * | 2019-08-29 | 2021-04-09 | 淮北师范大学 | Method for constructing peanut cotyledon regeneration system |
| CN110809935A (en) * | 2019-11-08 | 2020-02-21 | 广东省农业科学院果树研究所 | Method for improving germination rate of banana seeds |
| CN113040052A (en) * | 2021-04-22 | 2021-06-29 | 大连民族大学 | Method for quickly constructing peanut regeneration system by taking young leaves as explants |
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| CN101960988B (en) | 2012-07-04 |
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