Plant genetic transformation is a pivotal and essential step in modifying important agronomic traits using biotechnological tools, which primarily depend on the efficacy of transgene delivery and the plant regeneration system. Over the years, advancements in the development of delivery methods and regeneration systems have contributed to plant engineering and molecular breeding. Recent studies have demonstrated that the efficiency of plant transformation can be improved by simultaneously delivering meristem-developmental regulators, utilizing virus-mediated gene editing, and executing non-sterile in planta manipulations. Efficient genetic delivery and non-tissue culture regeneration systems are gradually being developed. This review summarizes diverse delivery strategies and in planta regeneration techniques aimed at improving the efficiency of plant genetic transformation. We also emphasize the integration and utilization of these emerging transgenic approaches for expediting future crop engineering.

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.

Agrobacterium rhizogenes (A. rhizogenes)-mediated transformation of hairy roots is a favored and flexible method for root gene functional analysis. However, the selection of transformants can be complex and time-consuming. Here, we describe our simplified method for the A. rhizogenes-mediated hairy root induction in young peanut shoots using an expression vector with RUBY for direct visual selection of transformants. Analyses verified that this method provides a high-efficiency gene transformation technique for peanut, with transformant frequencies between 46.2 and 73.7%. To test the utility of this method in gene functional analyses, it was used to overexpress AhLRX6 in hairy roots and we present our preliminary results indicating the production of thicker cells walls in root tips relative to the WT.

Mitoviral-derived sequences are frequently detected in plant genomes, encoding an RNA-dependent RNA polymerase (RdRp). These sequences share many similarities with mitoviruses that are known to commonly infect plant mitochondria. However, the functional characterization of nuclear-encoded mitoviral-RdRp remains unclear. This study elucidates the critical role of mRdRp (AT2G07749) in maintaining mitochondrial homeostasis and embryo viability, highlighting the dual role of viral-derived genes in plant development and stress response. Phylogenetic analysis reveals that mRdRp shares 96.8% identity with the mitoviral RdRp encoded by mitochondrial-genomes, suggesting that this nuclear mRdRp gene originated from horizontal transfer events following ancestral plant-mitovirus infections. To dissect mRdRp function, we generated a mRdRp knockout mutant via CRISPR-Cas9 or knockdown mutant by RNA interference (RNAi). These mRdRp mutants exhibited severe developmental defects, including dwarfism, embryo lethality, and sterility. Phenotypic assays further showed that mRdRp mutants displayed heightened susceptibility to ABA and rotenone, indicating impaired adaptive capacity to both hormonal and metabolic stress. Loss of mRdRp led to fragmented mitochondrial networks and a significant reduction in mitochondrial abundance in both leaf protoplasts and root meristematic cells. Additionally, mitochondrial-derived small RNA (sRNA) aberrantly accumulated in mRdRp mutants, which potentially disrupts endogenous RNA-silencing pathways that rely on sRNA-mediated gene regulation. Collectively, these results provide mechanistic insights into the function integration of a virus-derived gene into plant cellular networks, advancing our understanding of host–virus coevolution and the role of horizontally transferred viral genes in shaping plant physiology.