CN1169427C - Soybean transgenic plant in-situ fasciculated bud regeneration culture method - Google Patents
Soybean transgenic plant in-situ fasciculated bud regeneration culture method Download PDFInfo
- Publication number
- CN1169427C CN1169427C CNB021507821A CN02150782A CN1169427C CN 1169427 C CN1169427 C CN 1169427C CN B021507821 A CNB021507821 A CN B021507821A CN 02150782 A CN02150782 A CN 02150782A CN 1169427 C CN1169427 C CN 1169427C
- Authority
- CN
- China
- Prior art keywords
- msb
- medium
- explant
- bud
- days
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Landscapes
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The present invention relates to a regeneration culture method for in-situ fasciculated buds of transgenic soybean plants, which belongs to the technical field of biology and modern agriculture. The present invention adopts a mode for transforming axillary bud meristem by agrobacterium to remove the growing points of top parts of germinating aseptic seedlings of soybeans, two cotyledons are retained, and the infection and the co-culture of agrobacterium are carried out. After sterilization, sulfuric acid kanamycin is added in a bud extending culture medium and a rooting culture medium to achieve sieving to obtain a great amount of regeneration seedlings and soybean plants converted. The present invention utilizes a mode for transforming axillary bud meristem by agrobacterium, and different culture mediums are used indifferent growth periods to generate cespitose indefinite buds. The present invention has the advantages of fast growth of buds, few closed-top buds, robust plants, little regeneration difference among various varieties, short cultivation time, high conversion rate, and eliminates difference among genotypes of seeds to realize broad spectrum.
Description
Technical field
The present invention relates to a kind of soybean transgene plant regeneration cultural method, particularly a kind of soybean transgene plant original position shoot regeneration cultural method of growing thickly belongs to biotechnology and modern agricultural technology field.
Background technology
The success that plant gene transforms, depend on and whether have good receptor system, promptly whether have the stronger vegetable regeneration capacity and the conversion ratio of high frequency, find by literature search, Zhao Huiwu etc. are " moving towards the molecular biology of plants of 21 century " 446～447 pages, write articles " progress of plant genetic transformation technology ", the regeneration competent cell that this article proposes will to set up in Plant Transformation tissue culturing system will provide a large amount of foreign genes to be imported into, should select the converting material source easily, the tissue culturing system that the group training time is short.Too low with this method for transformation efficient of particle gun, have only about 0.3%.Also mentioned the person and use cotyledon for the 5th page in the book, cotyledonary node etc. are as explant, and the soybean transgene plant is succeedd.But owing to lack a regeneration plant system efficiently, the improvement of genes of soybean still is subjected to great restriction.
Summary of the invention
The objective of the invention is to overcome the conversion ratio of the low and low frequency of existing soybean plant strain regeneration capacity, a kind of soybean transgene plant original position shoot regeneration cultural method of growing thickly is provided, make it solve soybean regrowth and the few difficult point of conversion seedling in biotechnology, it is many to reach bud point, the regrowth rate is strong, by certain selection pressure and specific medium culture, form the effect of the transformed plant of high frequency.
The present invention is achieved through the following technical solutions, the present invention adopts the merismatic mode of Agrobacterium-mediated Transformation axillalry bud, remove the top growing point of the soybean aseptic seedling of sprouting, keep two cotyledons, and cause top part epidermis to break and hinder, carry out agroinfection and common cultivation, after the degerming, screen with a certain amount of kanamycin sulfate adding bud elongation medium and root media, the indefinite bud that impels its generation to grow thickly, blastogenesis is long fast, and the bud that binds is few, the plant strain growth stalwartness, various regeneration differences are little, incubation time shortens, and conversion ratio improves, and elimination seed cdna type differences reaches broad spectrum activity.Obtain the more regrowth and the soybean plant strain of conversion.
Below the inventive method is further described, method step is as follows:
(1) seed is through sterilization, be placed on MSB+6-BA (6-benzyladenine) 0.4mg/L, PH5.8, sprouted 3-5 days on the medium, remove terminal bud and make two cotyledons of wound reservation, obtain explant, be placed on MSB+6-BA10mg/L+IBA (indolebutyric acid) 0.3mg/L, PH5.8, medium cultivated one day in advance.
(2) use and to have B.t. (bacillus thuringiensis gene), gna (Galanthus Nivalis Agglutinin (GNA) gene) or pta (pinellia agglutinin gene) gene agroinfection explant 10 minutes, be placed on MSB+6-BA1.0mg/L+IBA0.2mg/L, PH5.8, medium was cultivated 3-4 days in advance, taking-up is placed on MSB+6-BA0.5mg/L+cb (cephalosporin) 500mg/L, PH5.8, explant degerming 2-3 week.
(3) explant after the degerming is placed on MSB+KM (kanamycin sulfate) 80mg/L, PH5.8, and on the medium, per 2 weeks are changed a subculture, screen to indefinite bud occurring, excise cotyledon when being elongated to 1-2cm, and are elongated to 3-4cm.
(4) downcut the indefinite bud on the explant and be placed on 1/2MSB+KM50-80mg/L, PH5.8 on the medium, transformed the bud root induction 15-20 days, was transplanted to big basin to obtaining the seed that transformed plant produces.
The present invention has prominent matter characteristics and marked improvement, compare with explants cultivation soybean transgene regeneration plants such as selecting cotyledon, cotyledonary node for use, it is long fast and the bud that binds is few that the present invention can produce the indefinite bud of growing thickly, blastogenesis, and these regenerated adventitious buds can easier elongation, and plant has higher conversion ratio, incubation time 80-110 days, shorten 20-30 days, compare with particle gun, conversion ratio increases substantially especially, and can eliminate difference between the soya seeds genotype, reach broad spectrum activity.The method can be used for soybean changes range gene, can obtain the transformed soybean plant of upper frequency fast.
Embodiment
Provide following three embodiment in conjunction with content of the present invention:
Embodiment 1
The experiment kind is rich 35 for closing, respectively 50;
Seed is placed on MSB+6-BA0.4mg/L through sterilization, and PH5.8 sprouted 3 days on the medium, removes terminal bud and make wound to keep two cotyledons, obtains explant, is placed on MSB+6-BA10mg/L+IBA0.3mg/L, and PH5.8, medium cultivated one day in advance; Infection has B.t. gene Agrobacterium 10 minutes (OD600nm0.5), is placed on MSB+6-BA1.0mg/L+IBA0.2mg/L, and PH5.8, medium cultivated 3 days in advance, takes out and is placed on MSB+6-BA0.5mg/L+cb500mg/L, PH5.8,2 weeks of explant degerming; Explant after the degerming is placed on MSB+KM80mg/L, PH5.8, and on the medium, per 2 weeks are changed a subculture, screen to indefinite bud occurring.Excision cotyledon when being elongated to 1cm, and be elongated to 3cm; Downcut the indefinite bud on the explant and be placed on 1/2MSB+KM50～80mg/L, PH5.8 on the medium, transformed the bud root induction 15 days, was transplanted to big basin to obtaining the seed that transformed plant produces.The soybean plant strain that obtains commentaries on classics B.t. gene closes rich No. 35 35 strains.Compare with the method for cotyledonary node regeneration, regeneration rate has improved 50%.
Embodiment 2
The test kind for close rich No. 35 50;
Seed is placed on MSB+6-BA0.4Mg/L through sterilization, and PH5.8 sprouted 4 days on the medium, removes terminal bud and make wound to keep two cotyledons, obtains explant, is placed on MSB+6-BA10mg/L+IBA0.3mg/L, and PH5.8, medium cultivated one day in advance; Infection has gna gene Agrobacterium 10 minutes (OD600nm0.5), is placed on MSB+6-BA1.0mg/L+IBA0.2mg/L, and PH5.8, medium cultivated 4 days in advance, takes out and is placed on MSB+6-BA0.5mg/L+cb500mg/L, PH5.8,3 weeks of explant degerming; Explant after the degerming is placed on MSB+KM80mg/L, PH5.8, and on the medium, per 2 weeks are changed a subculture, screen to indefinite bud occurring.Excision cotyledon when being elongated to 2cm, and be elongated to 4cn; Downcut the indefinite bud on the explant and be placed on 1/2MSB+KM50-80mg/L, PH5.8 on the medium, transformed the bud root induction 20 days, was transplanted to big basin to obtaining the seed that transformed plant produces.The soybean plant strain that obtains commentaries on classics gna gene closes rich No. 35 37 strains.Compare with the method for cotyledonary node regeneration, regeneration rate has improved 55%.
Embodiment 3
The test kind for close rich No. 35 50;
Seed is placed on MSB+6-BA0.4mg/L through sterilization, and PH5.8 sprouted 5 days on the medium, removes terminal bud and make wound to keep two cotyledons, obtains explant, is placed on MSB+6-BA10mg/L+IBA0.3mg/L, and PH5.8, medium cultivated one day in advance; Infection has pta gene Agrobacterium 10 minutes (OD600nm0.5), is placed on MSB+6-BA1.0mg/L+IBA0.2mg/L, and PH5.8, medium cultivated 4 days in advance, takes out and is placed on MSB+6-BA0.5mg/L+cb500mg/L, PH5.8,2.5 weeks of explant degerming; Explant after the degerming is placed on MSB+KM80mg/L, PH5.8, and on the medium, per 2 weeks are changed a subculture, screen to indefinite bud occurring.Excision cotyledon when being elongated to 2cm, and be elongated to 4cm; Downcut the indefinite bud on the explant and be placed on 1/2MSB+KM50-80mg/L, PH5.8 on the medium, transformed the bud root induction 18 days, was transplanted to big basin to obtaining the seed that transformed plant produces.The soybean plant strain that obtains commentaries on classics pta gene closes rich No. 35 30 strains.Compare with the method for cotyledonary node regeneration, regeneration rate has improved 60%.
Claims (2)
1, a kind of soybean transgene plant original position shoot regeneration cultural method of growing thickly, it is characterized in that, adopt the merismatic mode of Agrobacterium-mediated Transformation axillalry bud, removal is at MSB+6-BA0.4mg/L, the top growing point of the soybean aseptic seedling of sprouting on the medium of PH5.8, keep two cotyledons, and cause top part epidermis to break and hinder, obtain explant and be placed on MSB+6-BA10mg/L+IBA 0.3mg/L, the pre-cultivation after one day on the medium of PH5.8, carry out agroinfection, be placed on MSB+6-BA1.0mg/L+IBA0.2mg/L then, cultivated altogether in the medium of PH5.8 3～4 days, at MSB+6-BA0.5mg/L+cb 500mg/L, on the medium of PH5.8, after 2～3 weeks of explant degerming, at MSB+KM 80mg/L, the bud elongation medium of PH5.8 and 1/2MSB+KM 50～80mg/L, the root media of PH5.8 screens, and obtains the more regrowth and the soybean plant strain of conversion.
2, this soybean transgene plant original position according to claim 1 shoot regeneration cultural method of growing thickly is characterized in that method step is as follows:
(1) seed is placed on MSB+6-BA0.4mg/L through sterilization, and PH5.8 sprouted on the medium 3～5 days, removes terminal bud and make wound to keep two cotyledons, obtains explant, is placed on MSB+6-BA10mg/L+IBA0.3mg/L, and PH5.8, medium cultivated one day in advance;
(2) use the agroinfection explant 10 minutes have bacillus thuringiensis gene, Galanthus Nivalis Agglutinin (GNA) gene or pinellia agglutinin gene, be placed on MSB+6-BA1.0mg/L+IBA0.2mg/L, PH5.8, medium is pre-to be cultivated 3～4 days, taking-up is placed on MSB+6-BA0.5mg/L+cb 500mg/L, PH5.8,2～3 weeks of explant degerming;
(3) explant after the degerming is placed on MSB+KM 80mg/L, PH5.8, and on the medium, per 2 weeks are changed a subculture, screen to indefinite bud occurring, excise cotyledon when being elongated to 1～2cm, and are elongated to 3～4cm;
(4) downcut the indefinite bud on the explant and be placed on 1/2MSB+KM 50～80mg/L, PH5.8 on the medium, transformed the bud root induction 15～20 days, was transplanted to big basin to obtaining the seed that transformed plant produces.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB021507821A CN1169427C (en) | 2002-11-28 | 2002-11-28 | Soybean transgenic plant in-situ fasciculated bud regeneration culture method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB021507821A CN1169427C (en) | 2002-11-28 | 2002-11-28 | Soybean transgenic plant in-situ fasciculated bud regeneration culture method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1411706A CN1411706A (en) | 2003-04-23 |
| CN1169427C true CN1169427C (en) | 2004-10-06 |
Family
ID=4751834
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB021507821A Expired - Fee Related CN1169427C (en) | 2002-11-28 | 2002-11-28 | Soybean transgenic plant in-situ fasciculated bud regeneration culture method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1169427C (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101946623B (en) * | 2010-09-01 | 2012-01-04 | 中国农业科学院油料作物研究所 | Soybean non-tissue culture plant regeneration method and application thereof |
| CN102329816B (en) * | 2011-06-15 | 2012-12-26 | 北京未名凯拓作物设计中心有限公司 | Agrobacterium-mediated method for transferring soybean from top end of epicotyl of longitudinally-cut seedling |
-
2002
- 2002-11-28 CN CNB021507821A patent/CN1169427C/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| CN1411706A (en) | 2003-04-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CA2418056A1 (en) | Methods for stable transformation of plants | |
| WO2022135246A1 (en) | R gene for controlling matching of soybean-rhizobium, protein and use thereof | |
| CN100581352C (en) | Method for high frequency plant regenerating of tallow tree tissue culture adventitious bud | |
| CN102174562A (en) | Application of novel rooting method in soybean transgenic technology | |
| CN102154364A (en) | Method for agrobacterium tumefaciens-mediated genetic transformation of sugarcane | |
| CN113584072B (en) | Construction method of strawberry genetic transformation system | |
| CN101445808A (en) | Agrobacterium-mediated genetic transformation method with peanut seed domant bud hypocotyl as explant | |
| CN101144091B (en) | Method for obtaining switchgrass transgene plant | |
| CN101584297A (en) | Tissue culture and regenerated plant in vitro mycorrhization method for pinus massoniana | |
| CN108085334B (en) | Improved method for transforming barley microspore by agrobacterium | |
| CN100366145C (en) | Method for in-vitro breeding indirect regenerated plant of watermelon cotyledon | |
| CN102250943A (en) | In-vitro tissue culturing method for soybeans under mediation of agrobacterium tumefaciens | |
| CN110184293A (en) | A method of increasing phytomass or yield by improving photosynthetic efficiency | |
| CN109735538A (en) | A kind of carrier and its preparation method and application improving forest Strawberry Leaves regeneration efficiency | |
| CN101736028B (en) | Novel method of soybean genetic transformation independent of tissue culture by taking soybean cotyledonary node as receptor | |
| CN1169427C (en) | Soybean transgenic plant in-situ fasciculated bud regeneration culture method | |
| CN1926961A (en) | Method for increasing genetic conversion rate of transgenic soybean by using graft technology | |
| CN102450214A (en) | Screening and preserving method for Lolium L. embryogenic callus | |
| Barik et al. | High frequency in vitro regeneration of Lathyrus sativus L. | |
| CN101016544A (en) | Method of establishing winter jujube genetic conversion system for receptor by using stem tip | |
| CN115216490A (en) | Non-heading Chinese cabbage regeneration method in non-tissue culture mode | |
| CN115058381A (en) | Method for obtaining populus tremuloides haploid cell line | |
| CN113881698A (en) | Method for transforming barley microspore callus by utilizing agrobacterium | |
| CN112042532B (en) | Culture medium for inducing calluses to generate and application of culture medium in establishment of calluses regeneration system | |
| CN111621519A (en) | Genetic transformation method and application of succulent plant |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C19 | Lapse of patent right due to non-payment of the annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |