Systematic investigation and validation of peanut genetic transformation via the pollen tube injection method.

Genetic transformation is a pivotal approach in plant genetic engineering. Peanut (Arachis hypogaea L.) is an important oil and cash crop, but the stable genetic transformation of peanut is still difficult and inefficient. Recently, the pollen tube injection pathway has been shown to be effective for the genetic transformation of peanut. However, the poor reproducibility of this pathway is still controversial. In this study, the appropriate time and location of injection, along with transgenic screening, were systematically investigated in the pollen tube mediated peanut genetic transformation. Our findings revealed that Agrobacterium injections could be conducted within a time window of two to three hours preceding and succeeding the blooming process. Among the various selective markers evaluated, the Basta screening emerged as the most expedient, followed closely by the DsRed visual screening. According to resistance screening and molecular identification, the average transformation efficiency was 2.6% in the heritable transgenic progenies, which was more likely affected by individual operation by style cavity injection. Furthermore, the use of synergistic FT artificially regulated the blooming of peanuts under indoor conditions, facilitating operations involving keel petal injection and ultimately enhancing the genetic transformation efficiency. Thus, our study systematically validated the feasibility of peanut genetic transformation through an optimized pollen-tube injection technique without tissue culture, potentially guiding future advancements in peanut engineering and molecular breeding programs.