Measurement

In this project, by recording the growth cycle of Nicotiana benthamiana, we assisted the functional validation team in preparing for the optimal period of tobacco transient infection. To accurately determine the suitable injection period, we watered the plants with a fixed amount of water every other day while maintaining consistent environmental conditions. We measured the daily changes in leaf length and plant height after transplantation, with individual measurements taken for each leaf. Each group had three replicates, and two batches of seedlings were used in the experiments. The raw data can be found in the Excel spreadsheet.

For data analysis, we processed missing and outlier values using Excel. The preprocessed data was organized into line charts to visualize trends, and final effect plots were provided.

Our measurement tools offered the functional validation team more precise time points for processing, enabling accurate prediction of the optimal time window to prepare infection samples.

During the seedling cultivation period, we observed the following: 1. As shown in the raw data in the Excel sheet below, many leaves were still in the budding stage in the early period, so no growth records were taken; only significant growth was recorded later. 2. In the later growth records, some bottom leaves exhibited slowed growth, fluctuations in leaf length, and even wrinkling, which could be due to mild dehydration or the plant sacrificing nutrients from the lower leaves to support the growth of the upper leaves. 3. After more than four days of recording, the growth slope of newly emerged leaves was significantly steeper than that of the older leaves, indicating that the new leaves grew much faster than the older ones. This may have a beneficial effect on plant growth (apical dominance).

Due to the growth of the leaves, the gravity of the leaves will lower the height of the plant, and the contact with the plant during measurement may change the height of the plant, so the plant height here is unstable and there is a large fluctuation.

According to the chart, the plant height was generally slower in the early stage. The first and second batches both had large fluctuations in plant height between August 8 and August 14. Because the first batch and the second batch were not planted at the same time, the tobacco should be affected by environmental factors, and therefore, the instability of plant height makes plant height not a standard to measure the development of tobacco.

In plant experiments, we typically use Agrobacterium-mediated transient transformation of tobacco leaves for rapid functional validation. When infecting leaves, older leaves are generally not selected under non-specific conditions for the following reasons:

1. Cell Activity: Older leaves have lower cell activity and reduced metabolic activity, which are not conducive to the expression of foreign genes and reduce transformation efficiency.

2. Tissue Structure: Older leaves have thicker cell walls and a harder texture, which may hinder the transfer and absorption of foreign DNA, reducing transformation efficiency.

3. Health Condition: Older leaves may harbor more pathogens or be more affected by environmental stress, making them less healthy than younger leaves, which could impact the accuracy of experimental results.

4. Physiological State: Younger leaves are in a physiological state more suitable for gene expression and protein synthesis, enhancing the effectiveness of transient expression.

Therefore, selecting younger leaves for transient transformation experiments in tobacco can improve transformation efficiency and gene expression levels, ensuring the reliability and accuracy of the experimental results.

To determine the optimal injection period for younger leaves, our data analysis indicated that around the third week (approximately 16 days) after transplanting is suitable for preparing infection solutions and conducting bacterial culture shaking. We then performed transient injections at various growth stages of the tobacco seedlings, finding notable differences between early-stage new leaves and mature leaves (26 days after germination) at the injection stage.

Compared to younger leaves, mature tobacco leaves are fully expanded, have smoother surfaces, and clearly defined veins with fewer small veins, allowing a single injection to spread the infection solution over a larger area, often covering more than one-third of the leaf area, thereby reducing the number of injections needed. In contrast, immature leaves are not fully smooth, sometimes exhibiting uneven surfaces and numerous fine veins, making injection more challenging and resulting in a smaller spread area per injection, often not exceeding a 2 cm diameter circle. This necessitates multiple injections. Under these conditions, using overly young leaves for injection, such as when comparing luminescence differences, causes significant damage to the leaves and greatly affects the accuracy and stability of functional validation results.

Therefore, it is advisable to schedule bacterial culture shaking and infection solution preparation around the third week (approximately 16 days) after transplanting, with infection and phenotypic observation conducted one month from the initial sterilization treatment.

We can use the cultivation timeline of Nicotiana benthamiana shown in the diagram below to assess the optimal preparation time for infection and injection testing by the experimenters.

Around the third week (approximately 16 days) after transplantation, bacterial culture shaking can be scheduled, and the infection solution can be prepared. From the day of sterilization, the infection and phenotype observation should be conducted about one month later.