Effect of a suitable treatment period on the genetic transformation efficiency of the plant leaf disc method

BackgroundAgrobacterium tumefaciens-mediated leaf disc genetic transformation is an important way to achieve transgenics or gene editing. Ensuring stable and efficient genetic transformation is still an important problem in modern biology. It is assumed that the difference in the development status of genetic transformation cells of receptor materials is the main reason for the difference and instability of genetic transformation efficiency; the stable and efficient genetic transformation rate can be obtained by defining the appropriate treatment period of the receptor material and applying genetic transformation in a timely manner.ResultsBased on these assumptions, we studied and established an efficient and stable Agrobacterium-mediated plant transformation system with hybrid poplar (Populus alba x Populus glandulosa, 84 K) leaves, stem segments and tobacco leaves as the research objects. There were differences in the development process of leaf bud primordial cells from different explants, and the genetic transformation efficiency was significantly related to the cell development stage of the in vitro cultured materials. Among them, the genetic transformation rate of poplar and tobacco leaves was the highest on the 3rd and 2nd day of culture, reaching 86.6% and 57.3%, respectively. The genetic transformation rate of poplar stem segments was the highest on the 4th day of culture, reaching 77.8%. The best treatment period was from the development of leaf bud primordial cells to the S phase of the cell cycle. The number of cells detected using flow cytometry and 5-ethynyl-2MODIFIER LETTER PRIME-deoxyuridine (EdU) staining, the expression of cell cycle-related protein CDKB1; 2, CDKD1; 1, CYCA3; 4, CYCD1; 1, CYCD3; 2, CYCD6; 1, and CYCH; 1 of explants, and morphological changes of explants can be used as indicators to determine the appropriate treatment period for genetic transformation.ConclusionsOur study provides a new and universal set of methods and characteristics to identify the S phase of the cell cycle and apply genetic transformation treatments at the appropriate time. Our results are of great significance for improving the efficiency and stability of plant leaf disc genetic transformation.

Automated summary

This study investigates the relationship between the cell developmental stages and genetic transformation efficiency in different explants of hybrid poplar (Populus alba x Populus glandulosa, 84 K) leaves, stem segments, and tobacco leaves. The highest genetic transformation rates for poplar and tobacco leaves were observed on the 3rd and 2nd day of culture, reaching 86.6% and 57.3%, respectively. The highest genetic transformation rate for poplar stem segments was on the 4th day of culture, reaching 77.8%. By using indicators such as flow cytometry, cell cycle-related protein expression, and morphological changes of explants, the appropriate treatment period for genetic transformation was determined, improving the efficiency and stability of plant leaf disc genetic transformation.