In recent years, with the continuous improvement of transformation technology, the application of pollen tube pathway in agricultural molecular breeding has become more and more widespread. More than 20 crop varieties and strains, including rice, wheat, cotton, soybeans, and corn, have been obtained through the pollen tube pathway, establishing its position in direct transformation.
Principle
The basic principle of the pollen tube pathway-mediated transformation is to use the pollen tube pathway formed by pollen germination on the pistil stigma after plant pollination, inject exogenous DNA solution into the flower with a microsyringe, and carry the exogenous DNA into the embryo sac through the nucellar channel to achieve exogenous gene transfer. After double fertilization is completed in plants, the initial division of the fertilized egg cell requires sufficient accumulation of materials and energy. Cells at this stage do not yet have a complete cell wall and nuclear membrane system, and there is frequent exchange of materials inside and outside the cell. Exogenous fragments that penetrate into the embryo sac through the pollen tube pathway may enter the fertilized egg cells to achieve genetic transformation. When using the pollen tube pathway to introduce foreign genes, microinjection, stigmatic dripping, and pollen grain carrying methods are usually used. The flower organ of cotton is large, and microinjection is often used, that is, a microsyringe is used to inject genes into the fertilization chamber.
Compared with the vector transformation system and direct transformation system, the pollen tube pathway in the germplasm transformation system effectively utilizes the natural reproductive process of plants, avoids the problem of plant regeneration, and is simple, convenient and fast to operate. The limitation of this method is that it can only be used for genetic transformation of flowering plants, and the transformation can only be carried out during the flowering stage. With the continuous deepening of research, the technical system and related theories of pollen tube pathway will be more perfect.
Procedures
1. Preparation of donor DNA
The genomic DNA extracted from the donor plant or the plasmid DNA extracted from Escherichia coli and Agrobacterium are purified to prepare a DNA introduction solution in the form of genomic DNA or plasmid. Genomic DNA or plasmid DNA can also be digested to prepare a DNA introduction solution in the form of DNA fragments.
Preparation of DNA introduction solution: Dissolve the purified plant genomic DNA, plasmid DNA or enzyme-digested DNA fragments from E. coli and Agrobacterium in TE buffer or 1×SSC solution to prepare a DNA introduction solution. The DNA concentration is 50-100 µg/mL and set aside.
2. Preparation of transformation receptor materials
Normally developing flowers were used as transformation receptors. Select the flower buds that will bloom the next day for selfing, tie the front end of the finger-shaped corolla with a colored thin thread, and tie the other end of the thin thread to the bell handle as a harvest mark. After 20-24 hours of flowering, select the young ovaries with better fruiting branches and flower positions (generally the second to sixth fruit spurs) and the color of the flowers changing from white to pink as the transformation objects.
3. Donor DNA is introduced into the recipient
- Peeling the flowers: Before the donor DNA is introduced into the recipient, the corolla, stamens, stigmas, and styles must be removed, leaving only the exposed and intact young bolls and wrapping leaves. The styles should be broken off from the top of the young bolls so that the styles extending into the young bolls are flush with the tops of the young bolls. The specific operation method is to fix the young boll at the lower end with one hand, and grasp the corolla, including the stamens, stigma, and style, with the other hand, gently shake it left and right, and slowly pull it up. Be careful not to damage the epidermal layer of the ovary to avoid increasing the shedding rate.
- Donor DNA injection: Inhale the prepared DNA introduction solution into a 50 µL microsyringe. Hold the microsyringe with one hand and gently hold the young ovary after petal removal with the other hand. Insert the needle from the smoothed style along the longitudinal axis of the ovary to about 2/3 of the length of the ovary, and then retreat to about 1/3 of the length. Create a certain space in front of the needle to accommodate the donor DNA. Gently push the microsyringe to push the donor DNA solution into the fertilization chamber. Inject 5-10 µL of the DNA solution into each embryo. After injection, the seeds are quickly bagged and isolated until the seeds are mature.
4. Detection of transgenic plants
- Histochemical method. GUS gene expression was detected in calli and leaves according to the histochemical method. GUS detection of Kan-resistant callus: Cut the obtained Kan-resistant callus or leaves into small pieces, add them to the prepared X-gluc substrate solution, and incubate at 37°C overnight. Fix with FAA solution for more than 1 h, and decolorize with 70%, 90%, and 100% ethanol in sequence, and visually observe whether there is GUS gene expression on the surface and inside the callus.
- PCR identification. The genomic DNA of tobacco to-be-transgenic plants was extracted as a template. Design primers based on the selection marker gene or target gene sequence, and conduct PCR amplification detection using the total DNA of non-transgenic plants (negative control), empty strain transformed plants (negative control) and vector plasmid DNA (positive control) as controls to preliminarily identify whether the foreign gene has been integrated into tobacco genomic DNA.
- Southern blot analysis. The genomic DNA of tobacco to-be-transgenic plants was extracted and double-digested with restriction endonucleases. The processed DNA was electrophoresed on a 0.7%-0.9% agarose gel, and probe labeling, DNA transfer, membrane treatment, and hybridization operations were performed. Further identify whether the foreign gene is integrated into the genomic DNA of tobacco and the copy number of the foreign gene (control selection is the same as above).
- RT-PCR. Use the Trizol kit to extract the total RNA of tobacco to-be-transgenic plants, and use the reverse transcription PCR kit to perform RT reaction and PCR reaction on the total RNA according to the prescribed procedures. Further identify the expression of exogenous genes at the RNA level (control selection is the same as above).
- Northern blot analysis. Extract and purify the total RNA or mRNA of tobacco to-be-transgenic plants, conduct 1.2% (W/V) formaldehyde-denaturing agarose gel electrophoresis, and perform probe labeling, RNA transfer, membrane treatment, and hybridization operations. Further identify the expression of foreign genes in plant cells (control selection is the same as above).
Note:
- Before injection of donor DNA, peel the flowers gently to minimize damage and reduce the rate of shedding.
- The solutions and instruments (such as syringes) used in the experiment must be sterilized to avoid affecting the transformation efficiency.
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