CN114731953A - Construction of CIP149 efficient in-vitro regeneration system and stem genetic transformation system of diploid potatoes - Google Patents
Construction of CIP149 efficient in-vitro regeneration system and stem genetic transformation system of diploid potatoes Download PDFInfo
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- CN114731953A CN114731953A CN202210431330.0A CN202210431330A CN114731953A CN 114731953 A CN114731953 A CN 114731953A CN 202210431330 A CN202210431330 A CN 202210431330A CN 114731953 A CN114731953 A CN 114731953A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/002—Culture media for tissue culture
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
- C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
- C12N15/8205—Agrobacterium mediated transformation
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Abstract
The invention discloses a construction of a diploid potato CI P149 efficient in-vitro regeneration system and a stem genetic transformation system, which comprises the following steps: 1) the stem tips of the potato material CI P149 are cultured on MS30 basic culture medium; taking a stem section without a growth point as an explant and paving the explant on a P-MS20 plate culture medium for pre-culture; 2) activating a positive transformation strain of agrobacterium tumefaciens GV3101, shaking the strain to OD about 0.5, suspending the strain by using liquid MS20, adding AS, discarding the strain, placing the strain on a sterilized filter paper, sucking the strain to dryness, and then flatly paving the strain on a C-MS20 plate culture medium for culture; 3) uniformly replacing the explants co-cultured for two days on a culture medium for inducing callus; 4) the explants were uniformly changed to callus induction medium CI M for bud induction for 2 weeks, and then new bud induction medium SI M was changed every 2 weeks until budding. The genetic transformation system of the diploid potato CI P149 constructed by the invention has the advantages that the obtained callus is compact and dark green, the differentiation and the sprouting only need 2 months to 2 months, and the transformation efficiency is high.
Description
Technical Field
The invention relates to the technical field of genetic transformation, in particular to a high-efficiency in-vitro regeneration system of diploid potato CIP149 and construction of a stem genetic transformation system.
Background
The genetic transformation technique is a genetic improvement technique for introducing a target gene into a recipient plant genome by biological, chemical, physical or other methods to obtain stable inheritance and expression of the target gene, and is an important means for molecular breeding and gene function analysis. The success of plant gene transformation relies on a good transformation system to efficiently introduce and express foreign genes into recipient cells. The genetic transformation system of potato mediated by agrobacterium is mainly influenced by factors such as genotype, preculture, bacterial liquid concentration, infection time, co-culture and the like, and due to the heterogeneity of transformation receptors, verification and improvement are necessary according to actual conditions to obtain the optimal transformation conditions.
Advantages of diploid potatoes: the diploid potatoes account for 74 percent of total potato resources, are distributed more widely than common potato cultivars, and contain various resistance genes to diseases, insects and adverse environments and excellent quality character genes; meanwhile, the diploid potato has fewer chromosomes, is simple in heredity and is easy to analyze and carry out genetic operation. The development of the diploid potato resource excellent gene has important significance for solving the problems of narrow genetic background and poor gene bank of common cultivars (2 n-4 x-48), effectively improving the existing common cultivars and the like. Common potato cultivars have the ploidy level of the autotetraploid or partial autotetraploid and the difference of the Endosperm Balance Number (EBN), so that the common potato cultivars are difficult to have sexual hybridization with diploid potatoes, and the application of diploid germplasm resources in crop trait improvement is limited to a certain extent. Therefore, an efficient diploid potato tissue culture regeneration system is constructed, and the method utilizing genetic transformation is very important for the application of the diploid potato in crop improvement and plant genetic engineering.
Currently mature potato genetic transformation systems are mostly developed based on tetraploid materials, such as diclazy et al. Obvious genotype difference exists between the diploid potato material and tetraploid, and the conventional genetic transformation system is difficult to be suitable for the diploid material. It is therefore necessary to develop genetic transformation systems for specific diploid material.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides the construction of the CIP149 efficient in-vitro regeneration system and the stem genetic transformation system of the diploid potatoes, and the construction of the system has the advantages of high transformation efficiency, simple and convenient operation and the like, and has important significance for breeding practice and scientific research of the diploid potatoes.
In order to solve the technical problems, the invention adopts the following technical scheme:
the construction of the CIP149 efficient in-vitro regeneration system and the stem genetic transformation system of the diploid potato comprises the following steps:
1) the stem tip of the donor potato material CIP149 which is preserved in an aseptic way is cultured on an MS30 basal medium, the culture temperature is 23 +/-2 ℃, the humidity is 30-40%, the illumination intensity is 2000-; taking stem sections without growing points as explants, and laying the explants on a P-MS20 plate culture medium for preculture for 2 days, wherein each culture dish contains 100 explants;
2) activating a positive transformation strain of agrobacterium tumefaciens GV3101, shaking the strain to OD about 0.5, suspending the strain by liquid MS20, adding AS, impregnating the pre-cultured explant for 10min, discarding the bacterial solution, placing the explant on a sterilizing filter paper, sucking the bacterial solution, then flatly paving the explant on a C-MS20 plate culture medium, culturing for 2 days in a dark incubator at 28 ℃, and providing a proper growth environment for the agrobacterium tumefaciens and infecting stem segments more easily;
3) the explants which are co-cultured for two days are uniformly replaced on a culture medium for induced callus treatment, 20 stem segments are placed on each dish, the stem segments cannot be placed too many, the cut stem segments can secrete toxin to protect the stem segments from being damaged in the growth process of the culture medium, and too much toxin placed next to the stem segments can influence the growth of the stem segments;
4) and (3) uniformly replacing the explants processed in the step (3) after 2 weeks to a callus induction culture medium SIM for bud induction treatment, and then replacing a new SIM culture medium every 2 weeks until the explants bud.
As a preferable scheme of the invention, in the step 1), the bottle seedlings are propagated by 11-13 stem tips per bottle on average.
As a preferred embodiment of the present invention, in step 1), the selected explant is green in color and the plant tissue is compact.
As a preferable scheme of the invention, in the step 1), the basic culture medium consists of the following reagents in parts by weight:
as a preferable scheme of the invention, in the step 1), the stem section pre-culture medium consists of the following reagents in parts by weight:
as a preferable scheme of the invention, in the step 2), the C-MS20 is composed of the following reagents in parts by weight:
in a preferable embodiment of the invention, in step 3), the culture medium is CIM culture medium, and the CIM culture medium is composed of the following reagents in parts by weight:
as a preferable embodiment of the present invention, in step 4), the SIM medium is composed of the following reagents in parts by weight:
compared with the prior art, the invention has the following advantages:
1. according to the genetic transformation system of the diploid potato CIP149, the obtained callus is compact and dark green, only 2 months to 2 and a half months are needed for differentiation and sprouting, and the transformation efficiency is high (1900 stem segments are infected by the genetic transformation system, 105 transgenic seedlings are obtained, wherein 33 are positive plants); the used consumable reagents are all made in China, and the scientific research cost of the transgenosis is greatly reduced.
2. The construction of the system has the advantages of high transformation efficiency, simple and convenient operation and the like, and has important significance for breeding practice and scientific research of the diploid potatoes.
Drawings
FIG. 1 is a live view of C149 expanded propagation from 11 to 13 shoot tips;
FIG. 2 is a photograph showing the stem tip of 11-13 stem tips after C14920 days of propagation;
FIG. 3 is a live view of the pre-culture of C149 explants;
FIG. 4 is a view showing the condition that both ends of a part of C149 stem segments begin to swell after 2 days of co-culture;
FIG. 5 is a live image of C149 colonies transferred to Z2N0.1 medium (20 explants) after infection;
FIG. 6 is a photograph showing the live status of C149 stem segments in CIM medium for 2 weeks to induce callus;
FIG. 7 is a live picture of the beginning of budding of a stem segment cultured for 2 half-months on SIM;
FIG. 8 is a photograph showing the initiation of sprouting after 2 and a half months of culture in the C149 potato genetic transformation experiment;
FIG. 9 is a live picture of transgenic shoots that have undergone secondary rooting.
Detailed Description
The invention is described in further detail below with reference to the figures and the detailed description.
Materials: inoculating the single stem section of the CIP149 sterile test-tube plantlet on an MS basic culture medium, placing the MS basic culture medium in a tissue culture room, alternately culturing at the culture temperature of 23 +/-2 ℃, the humidity of 30-40 percent, the illumination intensity of 2000-. The stem of diploid potato C149 tissue culture seedling is used as explant, and the used medicines and reagents are analytically pure.
The construction method of the CIP149 efficient in-vitro regeneration system and the stem genetic transformation system of the diploid potatoes comprises the following steps:
1) the stem tips of the donor potato material CIP149 which is preserved in an aseptic mode are cultured on an MS30 basic culture medium (the average of 11-13 stem tips in each bottle of bottle seedlings is expanded and propagated), the culture temperature is 23 +/-2 ℃, the humidity is 30% -40%, the illumination intensity is 2000-. The stem section without growing points is taken as an explant and is laid on a P-MS20 plate medium for pre-culture for 2 days (the selected explant is green in color and compact in plant tissues), each culture dish contains 100 explants, the basic culture medium formula is described by MS30, and the pre-culture medium formula is described by P-MS 20. FIG. 1 is a live view of C149 expanded propagation from 11 to 13 shoot tips; FIG. 2 is a photograph showing the stem tip of 11-13 stem tips after C14920 days of propagation; FIG. 3 is a live view of the pre-culture of C149 explants.
MS30 medium: 1L of
Stem segment preculture medium (Z1N 2): 1L of
2) Activating a positive transformation strain of agrobacterium tumefaciens GV3101, shaking the strain to OD of about 0.5, suspending the strain by liquid MS20 (liquid), adding AS, dip-dyeing the pre-cultured explant for 10min, discarding the strain, placing the explant on a sterilizing filter paper, sucking the strain, and then flatly paving the explant on a C-MS20 plate culture medium to culture in the dark in an incubator at 28 ℃ for 2 days, thereby providing a proper growth environment for the agrobacterium tumefaciens and being capable of infecting stem sections more easily. FIG. 4 is a photograph showing the expansion of both ends of a part of the C149 stem segment after 2 days of co-culture.
Co-culture-MS20(C-MS20，Z1 N2)：1L
3) The explants which are co-cultured for two days are uniformly replaced on a culture medium for induced callus treatment, 20 stem segments are placed on each dish, the stem segments cannot be placed too many, the cut stem segments can secrete toxin to protect the stem segments from being damaged in the growth process of the culture medium, and too much nearby toxin placed on the stem segments can influence the growth of the stem segments. FIG. 5 is a live image of C149 colonies transferred to Z2N0.1 medium (20 explants) after infection; FIG. 6 is a photograph of the live status of C149 stem induced callus in CIM medium for 2 weeks.
CIM(Callus inducing medium,Z2N0.1):1L
4) And (3) uniformly replacing the explants processed in the step (3) after 2 weeks to a bud induction culture medium SIM for bud induction treatment, and then replacing a new SIM culture medium every two weeks until the explants bud. FIG. 7 is a live picture of the beginning of budding of a stem segment cultured for 2 half-months on SIM.
SIM(shoots inducing medium,Z1N0.01):1L
The high-efficiency regeneration system is utilized to carry out a potato genetic transformation experiment, 1900 CIP149 stem segments obtain 105 transgenic potato seedlings, 30 of which can root for the second time, and 33 positive seedlings are identified. FIG. 8 is a live picture of the initiation of sprouting after 2 and a half months of culture in C149 potato genetic transformation experiment, and FIG. 9 is a live picture of transgenic shoots which have undergone secondary rooting.
Finally, although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that various changes and modifications may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (8)
1. The construction of the CIP149 efficient in-vitro regeneration system and the stem genetic transformation system of the diploid potato is characterized by comprising the following steps of:
1) the stem tip of the donor potato material CIP149 which is preserved in an aseptic way is cultured on an MS30 basal medium, the culture temperature is 23 +/-2 ℃, the humidity is 30-40%, the illumination intensity is 2000-; taking stem sections without growing points as explants, and laying the explants on a P-MS20 plate culture medium for preculture for 2 days, wherein each culture dish contains 100 explants;
2) activating a positive transformation strain of agrobacterium tumefaciens GV3101, shaking the strain to OD about 0.5, suspending the strain by liquid MS20, adding AS, impregnating the pre-cultured explant for 10min, discarding the bacterial solution, placing the explant on a sterilizing filter paper, sucking the bacterial solution, then flatly paving the explant on a C-MS20 plate culture medium, culturing for 2 days in a dark incubator at 28 ℃, and providing a proper growth environment for the agrobacterium tumefaciens and infecting stem segments more easily;
3) the explants which are co-cultured for two days are uniformly replaced on a CIM culture medium for induced callus treatment, 20 stem segments are placed on each dish, the number of the stem segments cannot be increased, the cut stem segments can secrete toxin to protect the stem segments from being damaged in the growth process of the culture medium, and too much toxin placed close to the stem segments can influence the growth of the stem segments;
4) and (3) uniformly replacing the explants processed in the step (3) after 2 weeks to a callus induction culture medium SIM for bud induction treatment, and then replacing a new SIM culture medium every two weeks until the explants bud.
2. The construction of the diploid potato CIP149 efficient in vitro regeneration system and stem segment genetic transformation system according to claim 1, wherein: in the step 1), the bottle seedlings are propagated by 11-13 stem tips per bottle on average.
3. The construction of the diploid potato CIP149 efficient in vitro regeneration system and stem segment genetic transformation system according to claim 1, wherein: in the step 1), the selected explants are green in color, and the plant tissues are compact.
4. The construction of the diploid potato CIP149 efficient in-vitro regeneration system and the stem genetic transformation system according to claim 1, wherein: in the step 1), the basic culture medium consists of the following reagents in parts by weight:
5. the construction of the diploid potato CIP149 efficient in-vitro regeneration system and the stem genetic transformation system according to claim 1, wherein: in the step 1), the stem section pre-culture medium consists of the following reagents in parts by weight:
6. the construction of the diploid potato CIP149 efficient in vitro regeneration system and stem segment genetic transformation system according to claim 1, wherein: in step 2), C-MS20 is composed of the following reagents in parts by weight:
7. the construction of the diploid potato CIP149 efficient in-vitro regeneration system and the stem genetic transformation system according to claim 1, wherein: in the step 3), the culture medium is CIM culture medium, and the CIM culture medium is composed of the following reagents in parts by weight:
8. the construction of the diploid potato CIP149 efficient in-vitro regeneration system and the stem genetic transformation system according to claim 1, wherein: in step 4), the SIM culture medium consists of the following reagents in parts by weight:
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106636185A (en) * | 2016-11-14 | 2017-05-10 | 四川农业大学 | Culture medium and method for highly efficient regeneration and genetic transformation of somatic embryo of potato stems |
| CN110894539A (en) * | 2019-12-04 | 2020-03-20 | 云南师范大学 | Method for identifying self-compatibility of diploid potatoes |
| CN110938120A (en) * | 2019-12-04 | 2020-03-31 | 中国农业科学院农业基因组研究所 | StSCI protein for changing self-incompatibility of diploid potato material |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106636185A (en) * | 2016-11-14 | 2017-05-10 | 四川农业大学 | Culture medium and method for highly efficient regeneration and genetic transformation of somatic embryo of potato stems |
| CN110894539A (en) * | 2019-12-04 | 2020-03-20 | 云南师范大学 | Method for identifying self-compatibility of diploid potatoes |
| CN110938120A (en) * | 2019-12-04 | 2020-03-31 | 中国农业科学院农业基因组研究所 | StSCI protein for changing self-incompatibility of diploid potato material |
Non-Patent Citations (1)
| Title |
|---|
| 叶明旺等: "二倍体栽培马铃薯高效遗传转化体系的建立", 《中国农业科学》 * |
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Application publication date: 20220712 |