CN114921490B - Genetic transformation method for agrobacterium-mediated white clover callus - Google Patents
Genetic transformation method for agrobacterium-mediated white clover callus Download PDFInfo
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Abstract
The invention discloses an agrobacterium-mediated white clover callus genetic transformation method, which comprises the following steps: (1) establishing a genetic transformation receptor; (2) preparing agrobacterium engineering bacteria liquid; (3) infection and co-cultivation; (4) screening and culturing the resistant callus; (5) differentiation induction of the resistant callus; (6) rooting induction of the resistant buds; and (7) identifying the resistant plant molecules. The agrobacterium-mediated genetic transformation regeneration system provided by the invention is simple and easy to operate, has higher callus transformation rate, provides materials for genetic engineering research and molecular breeding of the white clover, and has scientific research value, economic value and ecological value.
Description
Technical Field
The invention belongs to the technical field of biological genetic transformation, and particularly relates to an agrobacterium-mediated white clover callus genetic transformation method.
Background
The Trifolium repens L.is a leguminous grass seed which is widely planted in the world, has the characteristics of high yield, excellent quality, high feeding value, preference for various livestock and the like, and meanwhile, the root of the Trifolium repens is rich in a large amount of rhizobia, has strong nitrogen fixation capability and high nitrogen fixation amount, and is often used for supplementing and improving degraded natural grasslands of pastures. In addition, the white clover has high ornamental value, full flowers, strong coverage of the ground surface and creeping growth expansion capability, and is also a lawn ground cover plant widely planted at home and abroad. However, the occurrence of adverse environmental events such as extreme temperatures, floods, salt water, and drought is increasing, limiting the productivity and affecting the yield of white clover.
Development of transgenic white clover provides a basic approach to solving the limitations in growth or productivity while applying traditional breeding methods. Since the whole genome of Trifolium pratense has been obtained, efficient use of genomic information will be helpful for functional genomics research. Meanwhile, the identification of the functional genes with prospects in stress-tolerant regulation and control mechanisms has important significance. However, little progress has been made in developing efficient genetic transformation systems for white clover. Screening of suitable explants and appropriate genetic transformation procedures for genetic transformation of Trifolium pratense is a major challenge.
Non-patent document Gu Juntao et al, "Agrobacterium-mediated efficient genetic transformation of Trifolium pratense and regeneration of transgenic plants," grass industry journal 16.2 (2007): 6. A method for genetic transformation of Trifolium pratense established by Agrobacterium Ti plasmid mediation is disclosed.
Non-patent document Wang Dan et al, "research on establishing genetic transformation system of Trifolium pratense by Agrobacterium-seed soaking method," Chinese agricultural science and technology guide 1 (2009): 6. A genetic transformation system of Trifolium pratense mediated by Agrobacterium-seed soaking method is disclosed.
Disclosure of Invention
Based on the background, the invention provides an agrobacterium-mediated white clover callus genetic transformation method, which uses seedling root systems as explant materials to induce callus, and then uses agrobacterium to infect the callus to carry out genetic transformation. The agrobacterium-mediated genetic transformation regeneration system has the characteristics of simple method, easy operation and the like, and provides powerful support for the application of the genetic engineering technology in the white clover.
The aim of the invention is achieved by the following technical scheme.
In a first aspect, the invention provides a genetic transformation method for agrobacterium-mediated white clover callus, which is characterized by comprising the following steps:
(1) Establishment of genetic transformation receptors
Taking roots, cotyledons, hypocotyls, leaves or petioles of 3-4 days of white clover seedlings as explants, and performing induction culture in a callus induction culture medium to obtain embryogenic callus;
(2) Preparation of engineering bacteria liquid of agrobacterium
Streak activation of Agrobacterium with TrCML6 plasmid on LB solid Medium to OD 600 Values between 0.4 and 0.5, and re-suspended in the infestation liquid for infestation;
(3) Infection and co-cultivation
Placing the embryogenic callus obtained in the step (1) into an invasion solution, infecting for 25-30min at a low rotation speed, placing the infected callus into a co-culture medium, and co-culturing with agrobacterium for 3-4 days at 23-25 ℃ under a dark condition;
(4) Screening culture of resistant callus
Washing the co-cultured callus, placing the co-cultured callus in a resistant callus screening culture medium, and continuously inducing the resistant callus for 14-16 days under dark conditions;
(5) Differentiation induction of resistant callus
Transferring the resistant callus induced in the step (4) into a differentiation screening culture medium, and inducing the generation of resistant buds under the conditions of 23-25 ℃ and 16h illumination/8 h darkness and 4000lx light intensity;
(6) Rooting induction of resistant buds
Cutting off the resistant buds obtained by induction in the step (5), transferring the resistant buds to a rooting screening culture medium, and inducing to generate a white clover root system;
(7) Molecular characterization of resistant plants
Transplanting the white clover seedlings which root and have good leaf growth in the step (6) into soil, and verifying whether the transformation is successful or not by a PCR method.
Preferably, the induction culture conditions in the step (1) are as follows: culturing in dark at 23-25deg.C for 5-6 weeks once every 2 weeks.
In a specific embodiment of the invention, the root, cotyledon and hypocotyl explants described in step (1) are taken from germinated 4-day white clover seedlings, specifically: transversely cutting root system and hypocotyl into 2-3cm segments; the cotyledons were separated along the cotyledon midline, and each cotyledon was cut into 2-3 pieces.
The leaf and petiole explants are taken from 30-day-old white clover aseptic seedlings, and specifically comprise the following steps: the petiole is cut into 3-4mm petiole sections, and the individual leaves of the white clover leaf blade are cut into 3-4 parts.
In a most preferred embodiment of the invention, the explant is selected from the root of white clover seedlings.
The agrobacterium with TrCML6 plasmid described in step (2) is obtained by the following method:
a: recombinant TrCML6 gene is carried out on a plasmid vector skeleton, and screening and identification are carried out to obtain an over-expression recombinant plasmid vector;
b: and (3) transforming the over-expression recombinant plasmid vector into agrobacterium by adopting a freeze thawing method to obtain the agrobacterium with the TrCML6 plasmid.
In a specific embodiment of the invention, the agrobacterium strain is EHA105, having rifampicin resistance; the plasmid vector was pBI121, the T-DNA region of which carries the GUS gene and the kanamycin resistance npt-II gene.
The TrCML6 gene is a trefoil calmodulin-like protein TrCML6 gene, and the nucleotide sequence of the TrCML6 gene is shown as SEQ ID NO. 1.
The nucleotide sequence shown in SEQ ID NO.1 is specifically as follows:
ATGGGATAAACACAAAAACACAGCACATTTCACATTTCCTCAAAACAAAACAAACCAAACAACATCATGTGTCCTTCTGGCAGAACCCTCCGTCCACAACCTCCCACAACCGATTTCCGACCGGCATTCGACATTCTCGACACCGATTGCGACGGCAAAATAAGCCGAGACGATCTCCGTTCATTCTACGCAACCACCAGCGGCGAAGGCGTCTCTGCCGACGCAATCGGTGCCATGATGTCGGTTGCGGACACAAACATGGACGGATTTGTGGAATACGAGGAATTCGAGCGTGTTGTTAGTGGAAACAATGAAAAGAAACCGTTAGGATGTGGAGCCATGGAAGATGTGTTCAAGGTGATGGATAGAGATGGTGATGGTAAACTTAGTCATGAAGATTTGAAGAATTATATGAATTGGGCTGGTTTTGCTGCAACAGATGAAGAGATAAATGCTATGATTAAGCTTGGTGGTGGTGATCAAAACGGTGGCGTTAGCTTCGATGGTTTGATTCGTATATTAGCTCTTGATCATTTCGTCCCTGTTTATTGATTCATTAA ATTAATGATGATATATTATTATCT
the TrCML6 gene is a functional gene which is obtained by analysis and screening of chitosan exogenously applied transcriptome data on the basis of early experiments and has the function of improving drought tolerance of the white clover. The application of the TrCML6 gene nucleotide sequence and the TrCML6 gene coded amino acid sequence in improving the drought resistance of plants can be seen in the patent of the invention of the inventor filed on the same day under the name of 'white clover calmodulin-like protein TrCML6 gene and the application of the same day in drought resistance'.
The preparation method of the culture medium at each stage comprises the following steps:
the preparation method of the callus induction culture medium in the step (1) comprises the following steps: MS medium added 30g/L sucrose and 200mg/L calcium gluconate, 6-BA to a final concentration of 0.5mg/L, 2,4-D to a final concentration of 2mg/L, pH adjusted to 5.8, 8g/L agar, at 121 ℃ autoclaved for 15min, VC to a final concentration of 10. Mu.g/L.
The preparation method of the infection liquid in the step (2) comprises the following steps: MS medium added 30g/L sucrose, 0.5g/L hydrolyzed casein and 200mg/L calcium gluconate, 6-BA to a final concentration of 0.5mg/L, 2,4-D to a final concentration of 2mg/L, pH adjusted to 4.8, autoclaved at 121℃for 15min, VC to a final concentration of 10. Mu.g/L, and AS to a final concentration of 20mg/L.
The preparation method of the co-culture medium in the step (3) comprises the following steps: MS medium added 30g/L sucrose, 0.5g/L hydrolyzed casein and 200mg/L calcium gluconate, 6-BA to a final concentration of 0.5mg/L, 2,4-D to a final concentration of 2mg/L, pH adjusted to 4.8, 8g/L agar, autoclaved at 121℃for 15min, VC to a final concentration of 10. Mu.g/L, and AS to a final concentration of 20mg/L.
The preparation method of the callus screening culture medium in the step (4) comprises the following steps: MS medium added 30g/L sucrose and 200mg/L calcium gluconate, 6-BA to a final concentration of 0.5mg/L, 2,4-D to a final concentration of 2mg/L, pH adjusted to 5.8, 8g/L agar, at 121 ℃ autoclaved for 15min, VC to a final concentration of 10. Mu.g/L, kan to a final concentration of 50mg/L, and Cef to a final concentration of 250mg/L.
The preparation method of the differentiation screening culture medium in the step (5) comprises the following steps: MS medium added 30g/L sucrose and 200mg/L calcium gluconate, 6-BA to a final concentration of 1.5mg/L, NAA to a final concentration of 0.1mg/L, pH adjusted to 5.8, 8g/L agar, at 121 ℃ autoclaved for 15min, VC to a final concentration of 10. Mu.g/L, kan to a final concentration of 50mg/L, and Cef to a final concentration of 250mg/L.
The rooting screening culture medium preparation method in the step (6) comprises the following steps: 30g/L sucrose and 200mg/L calcium gluconate are added to a 1/2MS medium, NAA is added to a final concentration of 1mg/L, the pH value is adjusted to 5.8, 8g/L agar is added, the mixture is autoclaved at 121 ℃ for 15min, VC is added to a final concentration of 10 mu g/L, kan is added to a final concentration of 50mg/L, and Cef is added to a final concentration of 250mg/L.
The Kan antibiotics are antibiotics corresponding to NPTII resistance genes carried by a target plasmid pBI121 transferred by agrobacterium.
In a most preferred embodiment of the present invention, the agrobacterium-mediated white clover callus genetic transformation method comprises the steps of:
(1) Taking roots of the white clover seedlings growing for 3-4 days as explants, placing the explants in a callus induction culture medium, culturing the explants at 23 ℃ under dark conditions, and carrying out subculture once every 2 weeks, and obtaining embryogenic callus after 5-6 weeks of growth;
(2) Activating agrobacterium with TrCML6 plasmid to OD600 value of 0.4, re-suspending in the infection liquid, placing the embryogenic callus obtained in the step (1) in the infection liquid, infecting for 25min at low rotation speed, placing the infected callus in a co-culture medium, and co-culturing with the agrobacterium for 4 days under the dark condition at 23 ℃;
(3) Washing the co-cultured callus, placing the co-cultured callus in a resistant callus screening culture medium, and continuously inducing the generation of the resistant callus under dark conditions;
(4) After 2 weeks, transferring the resistant callus induced in the step (3) into a differentiation screening culture medium, and inducing the generation of resistant buds once every 2 weeks under the conditions of 23 ℃ and 16h illumination/8 h darkness and 4000lx light intensity;
(5) Cutting off the resistant buds obtained by induction in the step (4), transferring the resistant buds to a rooting screening culture medium, and inducing the generation of Bai Sanshe root systems;
(6) Transplanting the positive white clover seedlings which root and have good leaf growth in the step (5) into soil, and verifying whether the transformation is successful or not by a PCR method.
Specific primers are designed through npt-II gene sequences, and the sequences of the primers for PCR are as follows:
an upstream primer: 5'-GAGGCTATTCGGCTATGACTGG-3' (SEQ ID NO. 2)
A downstream primer: 5'-ATCGGGAGCGGCGATACCGTA-3' (SEQ ID NO. 3)
The white clover is a Latin' white clover variety.
The genetic transformation method of the white clover callus provided by the invention has the following technical advantages:
(1) The inventor screens out the root of the white clover as an explant to carry out callus induction, and the obtained embryogenic callus is the optimal genetic transformation receptor of the white clover;
(2) The inventor adopts agrobacterium engineering bacteria liquid with TrCML6 plasmid to carry out infection, and finally, transgenic white clover plants with good drought resistance effect can be obtained;
(3) The genetic transformation method provided by the invention is simple and easy to operate, has higher transformation rate of callus, provides materials for genetic engineering research and molecular breeding of the white clover, and has scientific research value, economic value and ecological value.
Drawings
FIG. 1 callus induction rate (A) and callus browning rate (B) of different explants
FIG. 2 root system induced embryogenic callus
FIG. 3 callus after 4 days of co-culture with Agrobacterium
FIG. 4 selection of resistant calli after 2 weeks of induction
FIG. 5 differentiation screening explants after induction for 2 weeks
FIG. 6 cluster buds obtained by differentiation screening
FIG. 7 rooting and screening to obtain full-plant white clover seedlings
FIG. 8 PCR identification of transgenic white clover (A) and GUS staining of transgenic white clover (B)
Detailed Description
The technical solutions of the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only some embodiments of the present invention, but not all. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Table 1 abbreviation table
The formula of the culture medium related by the invention is as follows:
callus induction medium: MS+0.5 mg/L6-BA+2 mg/L2, 4-D+10. Mu.g/L VC+8g/L agar+30 g/L sucrose+200 mg/L calcium gluconate, pH=5.8;
and (3) dyeing liquid: MS+0.5 mg/L6-BA+2 mg/L2, 4-D+20mg/L AS+10. Mu.g/L VC+30g/L sucrose+0.5 g/L hydrolyzed casein+200 mg/L calcium gluconate, pH=4.8;
co-culture medium: MS+0.5 mg/L6-BA+2 mg/L2, 4-D+20mg/L AS+10. Mu.g/L VC+8g/L agar+30 g/L sucrose+0.5 g/L hydrolyzed casein+200 mg/L calcium gluconate, pH=4.8;
resistant callus screening media: MS+0.5 mg/L6-BA+2 mg/L2, 4-D+10. Mu.g/L VC+50mg/L Kan+250mg/L Cef+8g/L agar+30 g/L sucrose+200 mg/L calcium gluconate, pH=5.8;
differentiation screening medium: MS+1.5 mg/L6-BA+0.1 mg/L NAA+10. Mu.g/L VC+50mg/L Kan+250mg/L Cef+8g/L agar+30 g/L sucrose+200 mg/L calcium gluconate, pH=5.8;
rooting and screening culture medium: 1/2MS+1mg/L NAA+10. Mu.g/L VC+50mg/L Kan+250mg/L Cef+8g/L agar+30 g/L sucrose+200 mg/L calcium gluconate, pH=5.8.
Plant screening optimization for white clover epigenetic transformation
Soaking seeds of Leptoradix Trifolium Pratentis in 75% alcohol for about 5min, and washing twice with sterile water; subsequently, the seeds were placed in 0.1% HgCl 2 Soaking in the solution for 10 minutes, and carrying out surface sterilization on the seeds. After sterilization, washing with sterile distilled water for 5-6 times. Subsequently, the white clover seeds were spread evenly on sterile filter paper and germinated for 4 days, or the seeds were transferred to MS medium for 30 days. During this time, the plants were kept at 23℃with a 16h light/8 h dark cycle at an illumination intensity of 3000lx. The obtained aseptic seedlings or aseptic seedlings are used as materials for cutting explants.
Root, cotyledon and hypocotyl explants were taken from germinated 4-day white clover seedlings, specifically manipulated: transversely cutting root system and hypocotyl into 2-3cm segments; the cotyledons were separated along the cotyledon midline, and each cotyledon was cut into 2-3 pieces.
The explants of leaves and petioles were taken from 30 day old white clover seedlings, specifically: the petiole is cut into 3-4mm petiole sections, and the individual leaves of the white clover leaf blade are cut into 3-4 parts.
The obtained sterile explant is inoculated to a callus induction culture medium, and the wound of the explant is clung to the culture medium during inoculation, and the explant is placed at 23 ℃ to induce callus under dark condition. To ensure the nutrients of the medium, the explants were subcultured every 2 weeks. The callus induction rate was counted after 2 weeks, and the callus browning rate was counted after 4 weeks.
When inoculated on callus induction medium, callus can be obtained from cotyledons, hypocotyls, roots, leaves and petiole explants of Trifolium repens. After induction for 2 weeks, the callus induction rate of each explant reaches more than 88%; however, after 4 weeks of induction, cotyledons, hypocotyls, leaves and petioles induced callus were extensively browned, whereas the browning rate of root-induced callus was only 15.3% (fig. 1). After 4 weeks of induction, under a split microscope, the root-induced callus surface can see a large number of proembryos or somatic embryos (FIG. 2), while other explants perform poorly.
The preparation method of the callus induction culture medium comprises the following steps: MS medium added 30g/L sucrose and 200mg/L calcium gluconate, 6-BA to a final concentration of 0.5mg/L, 2,4-D to a final concentration of 2mg/L, pH adjusted to 5.8, 8g/L agar, at 121 ℃ autoclaved for 15min, VC to a final concentration of 10ug/L.
Genetic transformation method of white three-root callus
(1) Root callus acquisition
Soaking seeds in 75% alcohol for about 5 minutes, and then washing twice with sterile water; subsequently, the seeds were placed in 0.1% HgCl 2 Soaking in the solution for 10 minutes, and carrying out surface sterilization on the seeds. After sterilization, washing with sterile distilled water for 5-6 times. Spreading the imbibition seeds in a culture dish containing sterile filter paper, and cutting the explant after rooting (about 3-4 d). Cutting seedling root system into root sections with length of 2-3mm by using a sterile scalpel, placing the root sections in a Trifolium pratense callus induction medium, and inducing root source callus under the dark condition at 23 ℃. To ensure the nutrient components of the culture medium, the culture medium is subcultured every 2 weeks andthe brown callus during the subculture was discarded. After the root system callus of the white clover is induced for 40 days, the pale yellow embryogenic callus with compact structure and particle shape is picked out for infection of agrobacterium.
(2) Preparation of agrobacterium engineering bacteria liquid
The specific reference name of the primer pair design and cloning method is 'the trefoil calmodulin-like protein TrCML6 gene and the application thereof in drought resistance', and the TrCML6 gene sequence with 584bp total sequence length is obtained.
According to the pBI121 vector sequence, primers with Xba I and Sac I restriction sites and homology arm bases respectively introduced into the 5' end are designed: pBI121-TrCML-F (SEQ ID NO. 4) and pBI121-TrCML-R (SEQ ID NO. 5). PCR amplification was performed on TrCML6 with high fidelity enzyme using "Latin" white clover cDNA as template. The pBI121 vector was double digested with Xba I and Sac I, and the digested fragments were recovered. Connecting a PCR fragment of TrCML6 with a linearized pBI121 vector fragment by using a seamless recombinase, transforming DH5 alpha escherichia coli competent cells by using a connection product, coating the competent cells on an LB solid resistance plate containing 50mg/L Kan, culturing overnight at 37 ℃, picking a colony PCR verification recombinant vector, and sequencing and verifying to obtain a plant over-expression recombinant vector which is named pBI121-TrCML6.
pBI121-TrCML-F：
agaacacgggggactctagaATGTGTCCTTCTGGCAGAACCC
pBI121-TrCML-R:
ggggaaattcgagctcTCAATAAACAGGGACGAAATGA
And (3) performing amplification culture on the colony subjected to sequencing verification, and extracting the pBI121-TrCML6 over-expression recombinant vector. Transforming recombinant plasmid into Agrobacterium EHA105 competent cells by freeze thawing method, shake-activating and culturing the transformed competent cells at 28deg.C at 200rpm for 4h, centrifuging at 5000rpm for 1min in a centrifuge to collect bacterial liquid, gently beating re-suspended bacterial mass, coating on YEB solid culture medium containing 50mg/L Kan and 10mg/L rifampicin (Rif) antibiotics, and culturing in a 28 deg.C incubator for 2-3 days. And (3) picking single colonies, shaking and propagating, and detecting positive clones by adopting bacterial liquid PCR.
Melting 50 μl of EHA105 Agrobacterium in ice at-80deg.C, adding 1ug of pBI121-TrCML6 over-expression recombinant vector, mixing, standing on ice for 30min, liquid nitrogen for 3 min, water bath at 37deg.C for 5min, and ice bath for 5min. Add 500. Mu.l of YEB liquid medium without antibiotics, shake culture at 28℃for 4 hours at 200rpm, collect the bacterial liquid by centrifugation at 5000rpm for one minute, gently blow the resuspended pellet and spread on YEB solid medium with Kan, rif antibiotics, invert and incubate in 28℃incubator for 2-3 days. The bacterial liquid PCR detects positive clone, namely agrobacterium EHA105 with TrCML6 plasmid, the positive bacterial liquid is split into 2ml centrifuge tubes, 50% glycerol is added into each tube, and the bacterial liquid is stored at-80 ℃.
(3) Agrobacterium infection and co-cultivation
The agrobacterium with TrCML6 plasmid is thawed, 2ml of agrobacterium liquid is taken and cultured overnight at 28 ℃ and 200r/min in YEB liquid culture medium containing Kan and Rif antibiotics. Taking 50ml of bacterial liquid, centrifuging for 10min at 8000r/min, pouring out supernatant, suspending the bacterial liquid in the infection liquid again, and measuring the OD600 value of the bacterial liquid to be 0.4.
Embryogenic callus was cut into 5mm pieces and then placed into an Agrobacterium-harboring pBI121, and shaken at 28℃and 60rpm for 25min. The callus attached with the infection solution was gently nipped with sterile forceps to co-culture medium, and co-cultured at 23℃under dark conditions for 4 days, and the co-cultured callus was as shown in FIG. 3.
The preparation method of the infection liquid comprises the following steps: MS medium is added with 30g/L sucrose, 0.5g/L hydrolyzed casein and 200mg/L calcium gluconate, 6-BA to a final concentration of 0.5mg/L, 2,4-D to a final concentration of 2mg/L, pH adjusted to 4.8, autoclaved at 121 ℃ for 15min, VC to a final concentration of 10ug/L, and AS to a final concentration of 20mg/L.
The preparation method of the co-culture medium comprises the following steps: MS medium added 30g/L sucrose, 0.5g/L hydrolyzed casein and 200mg/L calcium gluconate, 6-BA to a final concentration of 0.5mg/L, 2,4-D to a final concentration of 2mg/L, pH adjusted to 4.8, 8g/L agar, autoclaved at 121℃for 15min, VC to a final concentration of 10ug/L, and AS to a final concentration of 20mg/L.
(4) Resistant callus screening
After the co-culture is finished, the callus is washed 3 to 4 times by sterile water, agrobacterium attached to the surface of the callus is washed, and the callus is placed on sterile filter paper to suck the surface moisture. The dried calli were transferred to a callus screening medium and callus growth was induced for 2 weeks. Untransformed calli can brown and die in the screening medium, and the transformed calli can normally grow and proliferate. As shown in FIG. 4, the partially transformed tissue developed new calli.
The preparation method of the callus screening culture medium comprises the following steps: MS medium added 30g/L sucrose and 200mg/L calcium gluconate, 6-BA to a final concentration of 0.5mg/L, 2,4-D to a final concentration of 2mg/L, pH adjusted to 5.8, 8g/L agar, at 121 ℃ autoclaved for 15min, VC to a final concentration of 10ug/L, kan to a final concentration of 50mg/L, and Cef to a final concentration of 250mg/L.
(5) Differentiation induction of resistant callus
Transfer the non-brown and well-grown callus to a differentiation screening culture medium, and induce bud differentiation in a dark environment of 4000lx, 23 ℃ and 16h illumination/8 h. To ensure that the nutrient composition and resistance titer of the medium meet the induction and elongation growth of the buds, and resistance screening, subculture is performed every 2 weeks. Untransformed callus will not differentiate; alternatively, the bud point is yellow and dies during differentiation. After about 2 weeks, the calli began to differentiate (as shown in fig. 5). After 6 weeks, the explants had differentiated the aerial parts of the white clover seedlings and the surrounding untransformed calli turned brown or the shoots turned yellow (as shown in FIG. 6).
The preparation method of the differentiation screening culture medium comprises the following steps: MS medium added 30g/L sucrose and 200mg/L calcium gluconate, 6-BA to a final concentration of 1.5mg/L, NAA to a final concentration of 0.1mg/L, pH adjusted to 5.8, 8g/L agar, at 121 ℃ autoclaved for 15min, VC to a final concentration of 10ug/L, kan to a final concentration of 50mg/L, and Cef to a final concentration of 250mg/L.
(6) Rooting induction of resistant buds
The induced white clover shoots are cut off by a sterile scalpel (only one shoot is taken out of each callus) and transferred into a rooting medium, and the shoots are inserted into the rooting medium by about 4-5mm. The seedlings were induced to root at 23℃under 16h light/8 h darkness with 4000lx light intensity. To ensure that the nutrient components and resistance titers of the culture medium meet the requirements of elongation growth and resistance screening of roots, the culture medium is changed every 2 weeks. As shown in FIG. 7, the root system of transgenic seedlings can grow 8-10cm after 4 weeks of induction.
The preparation method of the rooting screening culture medium comprises the following steps: 30g/L sucrose and 200mg/L calcium gluconate are added to a 1/2MS medium, NAA is added to a final concentration of 1mg/L, the pH value is adjusted to 5.8, 8g/L agar is added, the mixture is autoclaved at 121 ℃ for 15min, VC is added to a final concentration of 10ug/L, kan is added to a final concentration of 50mg/L, and Cef is added to a final concentration of 250mg/L.
(7) Identification of transgenic seedlings
Transferring the rooted seedlings into a growth chamber, uncovering, pouring distilled water, hardening the seedlings for 1 week. Extracting rooted white clover seedling from the culture medium, cleaning, transplanting to completely wet nutrient soil, and continuously culturing and propagating for 4 weeks. And (3) taking leaf tissues and petiole tissues for DNA extraction, and designing a specific primer through an npt-II gene sequence after DNA extraction, wherein the total amplification length is 700bp. And (3) taking the extracted DNA of the seedling as a template, performing PCR reaction, and verifying the integration condition of the marker gene.
Primer sequence for PCR:
an upstream primer: 5'-GAGGCTATTCGGCTATGACTGG-3' (SEQ ID NO. 2)
A downstream primer: 5'-ATCGGGAGCGGCGATACCGTA-3' (SEQ ID NO. 3)
As shown in FIG. 8A, 7 seedlings of 11 obtained white clover seedlings were finally determined as transgenic white clover seedlings after PCR detection, and the positive rate of the transgenic plants was 63%. After GUS staining was performed on untransformed and transgenic plants, respectively, the leaves of the control plants were bleached to yellow-white, and the transgenic plants were blue (as shown in FIG. 8B).
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.
Sequence listing
<110> Sichuan university of agriculture
<120> A method for genetic transformation of Agrobacterium-mediated white clover callus
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Claims (2)
1. An agrobacterium-mediated white clover callus genetic transformation method, characterized in that the genetic transformation method comprises the following steps:
(1) Taking roots of 3-4 days of white clover seedlings as explants, placing the explants in a callus induction culture medium, culturing the explants at 23 ℃ under dark conditions, carrying out secondary culture every 2 weeks, and obtaining embryogenic callus after 5-6 weeks of growth, wherein the white clover is Latin Bai Sanshe;
(2) Activating agrobacterium with TrCML6 plasmid to OD600 value of 0.4, re-suspending in the infection liquid, placing the embryogenic callus obtained in the step (1) in the infection liquid, infecting for 25min at low rotation speed, placing the infected callus in a co-culture medium, and co-culturing with the agrobacterium for 4 days under the dark condition at 23 ℃;
(3) Washing the co-cultured callus, placing the co-cultured callus in a resistant callus screening culture medium, and continuously inducing the generation of the resistant callus under dark conditions;
(4) After 2 weeks, transferring the resistant callus induced in the step (3) into a differentiation screening culture medium, and inducing the generation of resistant buds once every 2 weeks under the conditions of 23 ℃ and 16h illumination/8 h darkness and 4000lx light intensity;
(5) Cutting off the resistant buds obtained by induction in the step (4), transferring the resistant buds to a rooting screening culture medium, and inducing the generation of Bai Sanshe root systems;
(6) Transplanting the positive white clover seedlings which root and have good leaf growth in the step (5) into soil, and verifying whether the transformation is successful or not by a PCR method, wherein the primer sequences for PCR are as follows:
an upstream primer: 5'-GAGGCTATTCGGCTATGACTGG-3'
A downstream primer: 5'-ATCGGGAGCGGCGATACCGTA-3';
the TrCML6 gene is a trefoil calmodulin-like protein TrCML6 gene, and the nucleotide sequence of the TrCML6 gene is shown as SEQ ID NO. 1;
the preparation method of the callus induction culture medium in the step (1) comprises the following steps: adding 30g/L sucrose and 200mg/L calcium gluconate into MS culture medium, adding 6-BA to a final concentration of 0.5mg/L, adding 2,4-D to a final concentration of 2mg/L, adjusting pH to 5.8, adding 8g/L agar, autoclaving at 121deg.C for 15min, and adding VC to a final concentration of 10 μg/L;
the agrobacterium with TrCML6 plasmid described in step (2) is obtained by the following method:
a: recombinant TrCML6 gene is carried out on a plasmid vector skeleton, and screening and identification are carried out to obtain an over-expression recombinant plasmid vector;
b: transforming the over-expression recombinant plasmid vector into agrobacterium by adopting a freeze thawing method to obtain agrobacterium with TrCML6 plasmid;
wherein the agrobacterium strain is EHA105, having rifampicin resistance; the plasmid vector was pBI121, the T-DNA region of which carries the GUS gene and the kanamycin resistance npt-II gene.
2. The genetic transformation method according to claim 1, wherein the medium preparation method at each stage is as follows:
the preparation method of the infection liquid in the step (2) comprises the following steps: MS medium is added with 30g/L sucrose, 0.5g/L hydrolyzed casein and 200mg/L calcium gluconate, 6-BA is added to a final concentration of 0.5mg/L, 2,4-D is added to a final concentration of 2mg/L, pH value is adjusted to 4.8, the mixture is autoclaved at 121 ℃ for 15min, VC is added to a final concentration of 10 mug/L, and AS is added to a final concentration of 20 mg/L;
the preparation method of the co-culture medium in the step (2) comprises the following steps: MS medium is added with 30g/L sucrose, 0.5g/L hydrolyzed casein and 200mg/L calcium gluconate, 6-BA is added to a final concentration of 0.5mg/L, 2,4-D is added to a final concentration of 2mg/L, the pH value is adjusted to 4.8, 8g/L agar is added, the mixture is autoclaved at 121 ℃ for 15min, VC is added to a final concentration of 10 mug/L, and AS is added to a final concentration of 20 mg/L;
the preparation method of the callus screening culture medium in the step (3) comprises the following steps: MS medium added 30g/L sucrose and 200mg/L calcium gluconate, 6-BA to a final concentration of 0.5mg/L, 2,4-D to a final concentration of 2mg/L, pH adjusted to 5.8, 8g/L agar, autoclaved at 121℃for 15min, VC to a final concentration of 10. Mu.g/L, kan to a final concentration of 50mg/L, and Cef to a final concentration of 250 mg/L;
the preparation method of the differentiation screening culture medium in the step (4) comprises the following steps: MS medium added with 30g/L sucrose and 200mg/L calcium gluconate, 6-BA to a final concentration of 1.5mg/L, NAA to a final concentration of 0.1mg/L, pH adjusted to 5.8, 8g/L agar, autoclaved at 121℃for 15min, VC to a final concentration of 10. Mu.g/L, kan to a final concentration of 50mg/L, and Cef to a final concentration of 250 mg/L;
the rooting screening culture medium preparation method in the step (5) comprises the following steps: 30g/L sucrose and 200mg/L calcium gluconate are added to a 1/2MS medium, NAA is added to a final concentration of 1mg/L, the pH value is adjusted to 5.8, 8g/L agar is added, the mixture is autoclaved at 121 ℃ for 15min, VC is added to a final concentration of 10 mug/L, kan is added to a final concentration of 50mg/L, and Cef is added to a final concentration of 250mg/L.
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