RAPID: A Simple and Efficient Method for Plant Transformation

On April 8, 2024, the team of Xu Liu and Xingliang Hou from the Chinese Academy of Sciences published a research paper titled "A simple and efficient in planta transformation method based on the active regeneration capacity of plants" in Plant Communications, which reported a genetic transformation method without tissue culture.

Research Background

The accuracy of genetic modification mainly depends on the transformation of plants, which is difficult to achieve in most plant species. At present, gene editing based on traditional transformation methods has only been successfully achieved in a few representative crops. Although some progress has been made in the latest research on plant genetic transformation technology, in order to overcome the technical limitations and challenges brought by plant diversity, a new transformation strategy that is easy to be universally applied to various plants is still needed.

Research Results

A simple and efficient sweet potato plant genetic transformation system was established

Due to the natural regeneration ability of plants, plants can reproduce from excised organs, so independent transformants can be obtained by plant regeneration instead of tissue culture. The authors chose sweet potatoes with strong asexual reproduction ability to develop a tissue-free transformation method.

The authors used a variety of initial methods, including immersion, vacuum infiltration, and injection, to transfer Agrobacterium tumefaciens carrying the GUS reporter gene into plant tissues such as leaves, stems, flowers, and roots. At the same time, the effects of different culture media on the transformation effect were also considered. Finally, an optimal transfer method was obtained: stem injection and transplanting to soil matrix. The specific operation points are: cut off healthy sweet potato stems with several nodes (i.e., with side branches and side buds), and inject upward at each node until the injection liquid seeps out from other pinholes or cut ends. Adventitious roots sprout spontaneously within 1 week after transplantation, and positive mutants are quickly detected by GUS reporter gene.

Continuous cultivation of stem cuttings resulted in the production of transgenic new leaves, side buds, and tubers in adventitious roots, and positive signals were also detected in these tissues. Independent transgenic plants were obtained by asexual reproduction of side buds or buds sprouting from tubers in the subsequent short period of time. In addition, the authors also detected the location of T-DNA insertion in the genome of transgenic plants by TAILPCR and determined that these plants represented stable and independent transformation lines. New lateral branches and tuber buds of positive transformants showed no residual Agrobacterium contamination. These findings preliminarily confirmed that an effective in planta transformation method was established by utilizing the active regeneration ability of sweet potato plants.

Figure 1. Operating procedure of the stem-injection delivery system. (Mei, et al., 2024)

Optimization of the plant transformation system

Further, in order to improve the transformation efficiency, the authors measured the transformation rate from three conditions: the transformation strain, the strain concentration, and whether the chemical additives SilwetL77 and acetosyringone were added, and finally selected the transformation system most suitable for sweet potato. This study describes the development of a new plant transformation method that does not require sterile working conditions. The authors named this method RAPID. After optimization in sweet potato, the transformation rate of this method was close to 40%, and the duration was shortened to only 1 month. This method is significantly better than the traditional tissue culture method of sweet potato.

The RAPID method can deliver multiple reporter vectors

Next, the authors tested the application of several reporter genes on the RAPID system.

To avoid the interference of sweet potato autofluorescence, the authors selected mScarlet fluorescent protein (excitation wavelength 569 nm, emission wavelength 593 nm) as the reporter protein for transformation. As expected, most of the positive new tissues identified by PCR showed obvious red fluorescence. In addition, strong fluorescence signals were maintained in the leaves of transgenic plants regenerated from the transformants, indicating that mScarlet is a suitable fluorescent reporter gene for studying sweet potato transformation. Then, taking advantage of the property that RUBY can produce visible red pigments in living plants, the RUBY reporter system was constructed. Similar to other reporters, positive sweet potato plants showed obvious red phenotypes.

Finally, to determine whether the RAPID system is compatible with gene editing tools, the authors used CRISPR-Cas9 to knock out the phytoene desaturase desaturase (PDS) homolog (g31261) of sweet potato. Loss of PDS function produces obvious albino phenotypes in a variety of plants. The results showed that the transgenic new shoots gradually developed an obvious albino phenotype, and the gene editing was successfully completed.

Taken together, these results show that RAPID is a reliable method that can be used for the delivery of a variety of reporter vectors and can be used with gene editing systems.

RAPID produces positive new tissues by inducing successful transformation of meristem cells

Since lateral buds and adventitious roots develop from meristem cells of phloem tissue, the authors speculated that the RAPID method might induce transformation of tissues with regeneration capacity. To test this hypothesis, the authors used mScarlet as a reporter gene to analyze transformed stem segments. Untreated plants in the control group showed autofluorescence in the epidermis of the stem. Transformants showed clear fluorescent signals on the inner cross-section of the stem, and further observation revealed that these signals were mainly localized in the meristem region of the phloem, including the cambium and endoderm. The distribution of these structures is associated with the differentiation of lateral buds and adventitious roots in sweet potato. Similarly, lateral buds and adventitious roots of regenerated transgenic offspring showed strong positive signals. These observations indicate that the RAPID method can induce transformation of the meristem region through the phloem of plants and can produce stable regenerated plants from lateral buds and tuber buds. The time from injection to the production of lateral buds and tuber buds was 3-4 weeks and 6-8 weeks, respectively. All positive cell lines obtained from tuber shoots were non-chimeric, supporting the view that the transgene originated from one or a few meristem cells. These results indicate that the RAPID method can generate stable transgenic regenerated plants.

Figure 2. Direct delivery of Agrobacterium to the phloem by the RAPID system. (Mei, et al., 2024)

RAPID is applicable to different plant species

To evaluate the applicability of the RAPID method in different genetic backgrounds, the authors first tested different sweet potato varieties to detect GFP signals. Strong green fluorescence was observed in peeled stems, roots, and dissected tubers of GFP transformants, indicating that the RAPID method can be used to transform sweet potato plants of different genetic backgrounds. Overall, the rapid method has a high transformation rate for sweet potato (12.5% ​​-37.5%). Similarly, the authors performed multiple transformations on thick vines and potatoes, respectively, and obtained similar results as above.

Conclusion

In theory, RAPID can be applied to all plant species that develop into independent plants through asexual reproduction. This study developed a simple and effective Agrobacterium-mediated transformation system, which overcomes the technical limitations of existing transformation strategies, has the advantages of not requiring tissue culture and special induction, reduces the possibility of gene mutations, and improves transformation efficiency and is time-consuming.

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Reference

1. Mei, G., et al. A simple and efficient in planta transformation method based on the active regeneration capacity of plants. Plant Commun. 2024, 5(4): 10082.