CN1605248A - Tissue culture method of haricot in situ cluster seedling - Google Patents
Tissue culture method of haricot in situ cluster seedling Download PDFInfo
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- CN1605248A CN1605248A CN 200410084302 CN200410084302A CN1605248A CN 1605248 A CN1605248 A CN 1605248A CN 200410084302 CN200410084302 CN 200410084302 CN 200410084302 A CN200410084302 A CN 200410084302A CN 1605248 A CN1605248 A CN 1605248A
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Abstract
The present invention is in-situ haricot cespitose bud tissue culturing process in modern agricultural technology. The process adopts regenerated haricot axillary bud meristem and includes the following steps: sterilizing haricot seed, growing bacteria-free haricot seeding in germination culture medium, eliminating the lower plumular axis and top growing points while maintaining two cotyledons, setting the explant in germination culture medium, cutting grown adventitious bud for culturing in rooting culture medium to obtain regenerated seedling with root system, transplanting to big pot and culturing to obtain the regenerated seedling. The present invention can promote the generation of cespitose adventitious bud, and said process is suitable for tissue culture of various haricot varieties to obtain tissue culture seedling fast.
Description
Technical field
The present invention relates to a kind of method of French beans tissue culture, particularly a kind of tissue culture method of haricot in situ cluster seedling is used for the modern agricultural technology field.
Background technology
Utilize the plain improvement of biotechnology crop to become an important technology approach of modern agriculture.And tissue culture is the basis of biotechnology.The success of tissue culture depends on whether have good regenerative system.The French beans Study on tissue culture is fewer.
Find by prior art documents, Cheng Linmei etc. are at " Chinese oil crop journal ", 1998 20 (2): 6～8, write articles " research of soybean different explants plant regeneration " this article the different tissues inductivity of different soybean varieties be studies show that hypocotyl＞epicotyl＞little true leaf＞rataria, regeneration frequency average out to 34% is up to 50%.Report mentions that soybean regeneration induction plant frequency is low, poor repeatability.The indefinite bud of soybean group training is few, and the low growth of breeding rate is slow, and the variety and genetype differences is big in cultivation.French beans are legumes, and the same tissue culture with legume has difficulty, and an efficient French beans regeneration plant system is the key problem in technology that utilizes the bio-technology improvement French beans.
Summary of the invention
The present invention is directed to disadvantages of background technology and defective, overcome the low problem of regeneration capacity in the French beans tissue culture, a kind of tissue culture method of haricot in situ cluster seedling is provided, by plant regeneration system efficiently, make its solution French beans indefinite bud in tissue culture few, breeding quantity is low, the slow and big difficult point of genotype differences of growth, the regeneration bud that reaches the French beans tissue culture is many, and makes the purpose of regeneration plant stalwartness by the cultivation of medium.
The present invention is achieved by the following technical solutions, and the present invention adopts the merismatic method of French beans regeneration axillalry bud, and the French beans seed is sterile-processed, is placed on and sprouts the French beans aseptic seedling on the germination medium; The soybean that sprouts is removed hypocotyl, top growing point with scalpel under anatomical lens, keep two cotyledons, and make certain wound, explant is placed on the medium that sprouts; Indefinite bud is grown up to downcut and is placed on the root media again, grows root system and obtains being transplanted to big basin behind the regrowth, and the French beans indefinite bud with this culture method can much be grown thickly obtains regrowth after cultivating.
Below the invention will be further described, method step is as follows:
(1) seed disinfection and sprouting are cultivated.The seed water cleans and to be placed in 70% the alcohol 25～35 seconds, taking-up is placed among 10% the liquor natrii hypochloritis 15～25 minutes, with sterile water wash 2～4 times, disinfection seed is placed on MSB+6-BA (6-benzyladenine) 0.1～1mg/L, pH5.8 sprouted 3 days～5 days for 23 ℃～25 ℃ on the germination medium.
(2) original position is grown thickly, and bud is sprouted and grown cultures.Sprout French beans and remove hypocotyl, take out top growing point, keeps two cotyledons, be placed on MSB+6-BA 2～4mg/L, pH5.8, maintenance is 23 ℃～27 ℃ on the medium that sprouts, and cultivates 26 days～30 days, grows to 2～4cm height to bud.
(3) original position grow thickly bud cultivation and take root.Downcut indefinite bud and be placed on MSB+IBA (indolebutyric acid) 0.1～0.5mg/L, pH5.8,23 ℃ of root inductions are 23 days～25 days on the root media, are transplanted to big basin, obtain regrowth after cultivating.
The present invention has substantive distinguishing features and marked improvement, compare with explants cultivation regeneration plants such as selecting cotyledon, cotyledonary node for use, it is long fast and the bud that binds is few, plant strain growth is healthy and strong, incubation time shortens, tissue culture regeneration difference is little between each kind that the present invention can produce the indefinite bud of growing thickly, blastogenesis, good reproducibility, set up the tissue culture that the haricot in situ cluster seedling high efficient regeneration system is suitable for various French beans kinds with the inventive method, and can obtain French beans fast and organize seedling.
Embodiment
Content in conjunction with the inventive method provides following three embodiment:
Embodiment 1
The test kind is selected French beans No. 1,50 for handing over;
The seed water cleans and to be placed in 70% alcohol 25 seconds, takes out 10% sodium hydrosulfide 15 minutes, and with sterile water wash 2 times, disinfection seed is placed on MSB+6-BA 0.1mg/L, and pH 5.8, sprout 5 days for 23 ℃ on the germination medium; The French beans sprouted at the growing point of removing terminal bud and lateral bud between hypocotyl, two cotyledons under the anatomical lens with scalpel, are kept two cotyledons, be placed on MSB+6-BA 2mg/L, pH 5.8, keep 23 ℃ of sproutings 30 days on the medium that sprouts, and are cultured to the 2cm height; Downcut indefinite bud and be placed on MSB+IBA 0.1mg/L, pH5.8,23 ℃ of root inductions of root media 25 days are transplanted to big basin and are obtained regrowth; Obtain to hand over and select No. 1 regeneration plant 356 strains of French beans.An indefinite bud 7-10 that generation is grown thickly/, test-tube plantlet cultivated 60 days, blastogenesis is long fast and that the bud that binds is few, plant strain growth is healthy and strong, regeneration difference is cultivated by kind inner tissue is little, regeneration induction plant frequency reaches 712%.
Embodiment 2
The test kind is No. 1, mud city, 50;
The seed water cleans and to be placed in 70% alcohol 30 seconds, takes out 10% sodium hydrosulfide 20 minutes, and with sterile water wash 3 times, disinfection seed is placed on MSB+6-BA 0.5mg/L, and pH 5.8, sprout 4 days for 24 ℃ on the germination medium; The French beans sprouted at the growing point of removing terminal bud and lateral bud between hypocotyl, two cotyledons under the anatomical lens with scalpel, are kept two cotyledons, be placed on MSB+6-BA 3mg/L, pH 5.8, keep 25 ℃ of sproutings 28 days on the medium that sprouts, and are cultured to the 3cm height; Downcut indefinite bud and be placed on MSB+IBA 0.3mg/L, pH5.8,23 ℃ of root inductions are 24 days on the root media, are transplanted to big basin and obtain regrowth; Obtain No. 1, mud city, regeneration plant 389 strains.An indefinite bud 7-10 that generation is grown thickly/, test-tube plantlet cultivated 56 days, blastogenesis is long fast and that the bud that binds is few, plant strain growth is healthy and strong, regeneration difference is cultivated by kind inner tissue is little, regeneration induction plant frequency reaches 778%.
Embodiment 3
The test kind is No. 2, mud city, 50;
The seed water cleans and to be placed in 70% alcohol 35 seconds, takes out and is placed on 10% sodium hydrosulfide 25 minutes, and with sterile water wash 3 times, disinfection seed is placed on MSB+6-BA 1mg/L, and pH 5.8, sprout 3 days for 25 ℃ on the germination medium; The French beans sprouted at the growing point of removing terminal bud and lateral bud between hypocotyl, two cotyledons under the anatomical lens with scalpel, are kept two cotyledons, be placed on MSB+6-BA 4mg/L, pH 5.8, keep 27 ℃ of sproutings 26 days on the medium that sprouts, and are cultured to the 4cm height; Downcut indefinite bud and be placed on MSB+IBA 0.5mg/L, pH 5.8, and 23 ℃ of root inductions are 23 days on the root media, are transplanted to big basin and obtain regrowth; Obtain No. 2 regeneration plant 373 strains in mud city.An indefinite bud 7-10 that generation is grown thickly/, test-tube plantlet cultivated 52 days, blastogenesis is long fast and that the bud that binds is few, plant strain growth is healthy and strong, regeneration difference is cultivated by kind inner tissue is little, regeneration induction plant frequency reaches 746%.
The present invention has substantive distinguishing features and marked improvement, compare with explants cultivation regeneration plants such as selecting cotyledon, cotyledonary node for use, the present invention can produce the indefinite bud 7-10 of growing thickly/, test-tube plantlet and cultivate 52-60 days, blastogenesis is soon long and the bud that binds is few, plant strain growth is healthy and strong, incubation time shortens 20-30 days, good reproducibility, tissue culture regeneration difference is little between each kind, and regeneration induction plant frequency reaches 712%-778%.Set up the French beans original position with the inventive method and be suitable for the tissue culture of various French beans kinds, and can obtain French beans fast and organize seedling from the high efficient regeneration system of sprouting.
Claims (3)
1, a kind of tissue culture method of haricot in situ cluster seedling, its characteristic is, adopt the merismatic method of French beans regeneration axillalry bud, the French beans seed is sterile-processed, is placed on to sprout the French beans aseptic seedling on the germination medium, remove hypocotyl, top growing point, keep two cotyledons, explant is placed on the medium that sprouts, indefinite bud is grown up to downcut and is placed on the root media again, grow root system and obtain being transplanted to big basin behind the regrowth, after cultivating, obtain regrowth.
2, tissue culture method of haricot in situ cluster seedling according to claim 1 is characterized in that, further limits as follows by step to it:
(1) the seed water cleans and to be placed in 70% the alcohol 25～35 seconds, taking-up is placed among 10% the liquor natrii hypochloritis 15～25 minutes, and with sterile water wash 2～4 times, disinfection seed is placed on MSB+6-BA0.1～1mg/L, pH5.8 sprouted 3 days～5 days for 23 ℃～25 ℃ on the germination medium;
(2) sprout French beans and remove hypocotyl, top growing point, keep two cotyledons, be placed on MSB+6-BA2～4mg/L, pH5.8 keeps 23 ℃～27 ℃ on the medium that sprouts, and cultivates 26 days～30 days to 2～4cm height;
(3) downcut indefinite bud and be placed on MSB+IBA0.1～0.5mg/L, pH5.8,23 ℃ of root inductions are 23 days～25 days on the root media, are transplanted to big basin, obtain regrowth after cultivating.
3, tissue culture method of haricot in situ cluster seedling according to claim 2, it is characterized in that, in the step (2), described sprouting French beans take out top growing point, and the French beans of be about to sprouting are removed the growing point of terminal bud and lateral bud between hypocotyl, two cotyledons with scalpel under anatomical lens.
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| Application Number | Priority Date | Filing Date | Title |
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| CN 200410084302 CN1605248A (en) | 2004-11-18 | 2004-11-18 | Tissue culture method of haricot in situ cluster seedling |
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| CN 200410084302 CN1605248A (en) | 2004-11-18 | 2004-11-18 | Tissue culture method of haricot in situ cluster seedling |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102057872A (en) * | 2010-11-10 | 2011-05-18 | 上海市浦东新区农业技术推广中心 | Regeneration culture method of whole cotyledonary joint of hyacinth bean |
| CN102150622A (en) * | 2011-04-28 | 2011-08-17 | 江苏省农业科学院 | Method for regenerating plants by using adzuki bean tissues |
| CN104472352A (en) * | 2014-11-19 | 2015-04-01 | 天津科润农业科技股份有限公司 | Establishment method of direct regeneration system of phaseolus vulgaris |
-
2004
- 2004-11-18 CN CN 200410084302 patent/CN1605248A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102057872A (en) * | 2010-11-10 | 2011-05-18 | 上海市浦东新区农业技术推广中心 | Regeneration culture method of whole cotyledonary joint of hyacinth bean |
| CN102150622A (en) * | 2011-04-28 | 2011-08-17 | 江苏省农业科学院 | Method for regenerating plants by using adzuki bean tissues |
| CN104472352A (en) * | 2014-11-19 | 2015-04-01 | 天津科润农业科技股份有限公司 | Establishment method of direct regeneration system of phaseolus vulgaris |
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