A simple and efficient in planta transformation method based on the active regeneration capacity of plants

Abstract

Plant genetic transformation strategies serve as essential tools for the genetic engineering and advanced molecular breeding of plants. However, the complicated operational protocols and low efficiency of current transformation strategies restrict the genetic modification of most plant species. This paper describes the development of the regenerative activity-dependent in planta injection delivery (RAPID) method based on the active regeneration capacity of plants. In this method, Agrobacterium tumefaciens is delivered to plant meristems via injection to induce transfected nascent tissues. Stable transgenic plants can be obtained by subsequent vegetative propagation of the positive nascent tissues. The method was successfully used for transformation of plants with strong regeneration capacity, including different genotypes of sweet potato (Ipomoea batatas), potato (Solanum tuberosum), and bayhops (Ipomoea pes-caprae). Compared with traditional transformation methods, RAPID has a much higher transformation efficiency and shorter duration, and it does not require tissue culture procedures. The RAPID method therefore overcomes the limitations of traditional methods to enable rapid in planta transformation and can be potentially applied to a wide range of plant species that are capable of active regeneration.

Keywords: RAPID; active regeneration; bayhops; plant genetic transformation; potato; sweet potato.

Figure 1

Operating procedure of the stem-injection delivery system. (A) Operating procedure of the stem-injection delivery system. Healthy stems with several nodes were excised from sweet potato plants, and each node was injected upward until the solution oozed from the adjacent pinholes and excised end. The injected stems were planted in soil substrate. (B) Evaluation of the transformation of nascent roots. Adventitious roots sprouted spontaneously in the soil within 1 week (below the yellow line) and were collected for GUS staining. Scale bars, 1 cm (left), 0.5 cm (middle), and 0.5 mm (right). (C) Vegetative propagation of positive tissues. Independent transgenic plants were obtained by vegetative propagation of positive lateral shoots or from buds that sprouted from positive tubers. Positive rate = average (positive/total) × 100%.

Figure 2

Optimization of the stem-injection delivery system. (A) Transformation efficiency with different Agrobacterium strains. (B) Transformation efficiency with Agrobacterium strains at different OD values. (C) Transformation efficiency after addition of different additives. Positive rate = [average (positive roots/total number of roots per positive plant)] × (number of positive plants/total number of injected plants) × 100%. (D) Bulk selection of transgenic materials based on phosphinothricin (Basta) resistance. Phenotypes of injected plants before and after spraying Basta are shown. (E) Morphology of the positive plants and negative control plants 72 h after Basta spraying. The bar chart shows the positive rates based on Basta resistance and on genotyping of the screened plants. Positive rate = number of positive plants/total number of plants (%). The data are presented as the mean ± standard deviation of 10 biological replicates (two-tailed Student’s t-test; NS, no significance; P > 0.05). Scale bars, 5 cm in (D) and 5 cm in (E).

Figure 3

Applicability of reporter vectors and gene editing tools in RAPID. (A) Applicability of the mScarlet reporter in RAPID transformation. Scale bar, 0.5 cm. The table shows results obtained from three independent replicates. BL, bright light; FL, fluorescent light. (B) Applicability of the RUBY reporter in RAPID transformation. Scale bar, 2 cm. The table shows results obtained from three independent replicates. (C) Applicability of the CRISPR–Cas9 tool in transformation following PDS knockout. Scale bar, 2 cm. The table shows results obtained from three independent replicates. Vector schematics are shown on the right.

Figure 4

Direct delivery of Agrobacterium to the phloem by the RAPID system. (A) mScarlet fluorescence in a cross-section of sweet potato stem. The white arrow indicates the clear fluorescence signal in the transformant tissue. Scale bar, 0.5 mm; bright light, BL; fluorescent light, FL. (B) Histological observation of the mScarlet reporter signal. The areas within the yellow lines in the transverse (left) and longitudinal stem sections (right) depict the mScarlet signal in the transformant; scale bar, 0.1 mm. Vascular cell, vc; parenchymal cell, pc; cortex, cor; epidermis, ep. (C) Working model of the acquisition of transformed generations after direct transformation by the RAPID method.

Figure 5

Application of the RAPID system to other plant species. (A) Transformation of different sweet potato varieties using the RAPID method. The stem was peeled and observed under light and dark conditions (within the box). The arrow indicates the green fluorescence due to the GFP reporter (35S:GFP). Positive rate = number of positive plants/total number of plants (%). Scale bar, 2 cm; bright light, BL; fluorescent light, FL. (B) Transformation of bayhops. The white arrow indicates nascent shoots produced from the transformed stems. Leaves of transgenic shoots carrying the Bar gene (35S:Bar) retained their green color after phosphinothricin (Basta) treatment, but the wild-type leaves did not. Scale bars, 2 cm (left) and 1 cm (right). The table depicts the results obtained from three independent replicates. (C) Transformation of potato. Transgenic materials carrying the RUBY (DR5:RUBY) and mScarlet (35S:mScarlet) reporters exhibited positive phenotypes. Scale bars, 2 cm (upper) and 0.5 cm (lower). The table depicts the results obtained from three independent replicates.