An efficient Agrobacterium-mediated transient transformation system and its application in gene function elucidation in Paeonia lactiflora Pall

Abstract

Paeonia lactiflora Pall. is known as the king of herbaceous flowers with high ornamental and precious medicinal value. However, the lack of a stable genetic transformation system has greatly affected the research of gene function in P. lactiflora. The Agrobacterium-mediated transient gene expression is a powerful tool for the characterization of gene function in plants. In this study, the seedlings of P. lactiflora were used as the transformation receptor materials, and the efficient transient transformation system with a GUS reporter gene was successfully established by Agrobacterium harboring pCAMBIA1301. To optimize the system, we investigated the effects of germination time, Agrobacterium cell density, infection time, acetosyringone (AS) concentration, co-culture time, negative pressure intensity, Tween-20 concentration and different receptor materials on the transient transformation efficiency of P. lactiflora. The results showed that the highest transient transformation efficiency (93.3%) could be obtained when seedlings in 2-3 cm bud length were subjected to 12 h infection of resuspension solution comprising 1.2 OD600Agrobacterium, 200 μM AS and 0.01% Tween-20 under 10 of negative pressure intensity followed by 3 days of co-culture in darkness condition. This method is more suitable for the study of gene function in P. lactiflora. Subsequently, stress resistance genes PlGPAT, PlDHN2 and PlHD-Zip were used to verify the effectiveness of this transformation system. These results can provide critical information for identification of key genes in non-model plants, such as P. lactiflora, and promote the development of molecular biology research for P. lactiflora.

Transcriptional regulatory cascade of LcMYB71 and LcNAC73 affects low-temperature and drought stress response in Lonicera caerulea

“…found that there was no significant change in the expression of the gene after co-culturing young leaves of Populus alba for 3-5 days ( Song et al., 2019 ). We found in the experiment that the transformation efficiency of L. caerulea was stable after co-cultivation for 3-6 days, which is similar to previous research results ( Guan et al., 2022 ; Wu et al., 2022 ). The culture medium and co-transformation time used in this study differ slightly from the experimental methods used in the previous article due to the different plant materials, but the above transient genetic transformation systems achieved the goal of quickly identifying gene function and the regulatory relationship between genes.…”

“…If the infection time is too long, it will cause the explants to suffer from hypoxia and decay due to the toxicity of Agrobacterium ; The short duration of infection prevents successful transformation. The optimal conversion time for transient transformation of Paeonia lactiflora is 12 h ( Guan et al., 2022 ). The optimal time for transient transformation of lilies ( Lilium longiflorum ) is 5 h ( Wu et al., 2022 ).…”

An efficient screening system of disease-resistant genes from wild apple, Malus sieversii in response to Valsa mali pathogenic fungus

“…In contrast to other transient transformation methods, such as biolistic transformation and syringe infiltration, it is simple, quick, economical, and effective. In the last decade, the function of a growing number of genes in Tamarix hispida , Betula platyphylla, Morus alba , Populus trichocarpa , Withania somnifera and Paeonia lactiflora have been identified using this full seedlings soaking transient transformation method [ 15 – 17 , 21 , 30 , 43 , 46 , 49 , 54 , 58 , 71 , 72 , 74 ]. It also facilitates research on regulatory networks [ 23 , 24 , 59 , 65 ] and reverse chromatin immunoprecipitation technique [ 54 , 58 , 64 ].…”

Establishment of a Homologous Silencing System with Intact-Plant Infiltration and Minimized Operation for Studying Gene Function in Herbaceous Peonies

“…Previous research focusing on the construction of the VIGS system yielded optimal results and shed light on the transient transformation in geophytes [56,57]. Inspired by the cormlet and bulblet used as materials for VIGS to investigate gene regulation in bud growth transitions and corms dormancy in bulbous flowers, we established a homologous silencing system with intact-plant infiltration and minimized operation based on a prior study on VIGS system construction in herbaceous and tree peonies [44,58,59]. This established methodology is also applicable to more than 30 species of herbaceous peonies and can provide a valuable reference for all other geophytes.…”