Transient transformation meets gene function discovery: the strawberry fruit case

Liu

et al. 2016

Sci Rep

Trihelix genes play important roles in plant growth and development and responses to biotic and abiotic stresses. Here, we identified 56 full-length trihelix genes in Populus trichocarpa and classified them into five groups. Most genes within a given group had similar gene structures and conserved motifs. The trihelix genes were unequally distributed across 19 different linkage groups. Fifteen paralogous pairs were identified, 14 of which have undergone segmental duplication events. Promoter cis-element analysis indicated that most trihelix genes contain stress- or phytohormone-related cis-elements. The expression profiles of the trihelix genes suggest that they are primarily expressed in leaves and roots. Quantitative real-time reverse transcription polymerase chain reaction analysis indicated that members of the trihelix gene family are significantly induced in response to osmotic, abscisic acid, salicylic acid, methyl jasmonate and pathogen infection. PtrGT10 was identified as a target gene of miR172d, which is involved in the osmotic response. Repression of PtrGT10 could increase reactive oxygen species scavenging ability and decrease cell death. This study provides novel insights into the phylogenetic relationships and functions of the P. trichocarpa trihelix genes, which will aid future functional studies investigating the divergent roles of trihelix genes belonging to other species.

Section: Discussionmentioning

confidence: 97%

Comprehensive analysis of trihelix genes and their expression under biotic and abiotic stresses in Populus trichocarpa

Liu

et al. 2016

Sci Rep

“…Stable transformation is a labor-intensive low-throughput process. Comparatively, transient transformation is an easy and efficient method for gene transformation, which avoids the drawbacks of the stable transformation process, such as transformation efficiency, transformants selection, and regeneration [1]. Additionally, transient expression represents a rapid method to analyze the function of certain genes, and the analysis can be completed within a few days of transformation [2].…”

Section: Introductionmentioning

confidence: 99%

An Improved Syringe Agroinfiltration Protocol to Enhance Transformation Efficiency by Combinative Use of 5-Azacytidine, Ascorbate Acid and Tween-20

Zhao

Tan

Wen

et al. 2017

Plants

Syringe infiltration is an important transient transformation method that is widely used in many molecular studies. Owing to the wide use of syringe agroinfiltration, it is important and necessary to improve its transformation efficiency. Here, we studied the factors influencing the transformation efficiency of syringe agroinfiltration. The pCAMBIA1301 was transformed into Nicotiana benthamiana leaves for investigation. The effects of 5-azacytidine (AzaC), Ascorbate acid (ASC) and Tween-20 on transformation were studied. The β-glucuronidase (GUS) expression and GUS activity were respectively measured to determine the transformation efficiency. AzaC, ASC and Tween-20 all significantly affected the transformation efficiency of agroinfiltration, and the optimal concentrations of AzaC, ASC and Tween-20 for the transgene expression were identified. Our results showed that 20 μM AzaC, 0.56 mM ASC and 0.03% (v/v) Tween-20 is the optimal concentration that could significantly improve the transformation efficiency of agroinfiltration. Furthermore, a combined supplement of 20 μM AzaC, 0.56 mM ASC and 0.03% Tween-20 improves the expression of transgene better than any one factor alone, increasing the transgene expression by more than 6-fold. Thus, an optimized syringe agroinfiltration was developed here, which might be a powerful method in transient transformation analysis.

“…Strawberry (Fragaria), as a distinct member of Rosaceae, is not only an important fruit crop worldwidely, but is also regarded as a model for studying non-climacteric fruit ripening [1]. In the past years, a large of genes related to strawberry fruit ripening have been identi ed via traditional stable and transient transgenic systems in strawberry; however, more or less, the limitation is existed [2][3][4]. Thus, development of a rapid, e cient, and stable transformation system is required for strawberry research and breeding.…”

Section: Introductionmentioning

confidence: 99%

“…Such as, it takes at least 15 months from transformation to gaining transgenic fruits for gene function analysis in ripening [17,18]. In addition, although the transient transformation technology in strawberry fruits provides a rapid tool to study gene function in ripening, some limits are existent in fruit transient transgenic system [2,4].…”

Section: Introductionmentioning

confidence: 99%

Establishment of a Rapid and Stable Transgenic System by Agrobacterium-Mediated Transformation of the Germination Seeds in Diploid Strawberry.

Gu¹,

Shen²

2020

Preprint

Abstract BackgroundStrawberry (Fragaria) is regarded as a model plant for both Rosaceae and non-climacteric fruit ripening. Although much progress has been made in the identification of gene function using traditional, stable and transient genetic transformation systems in strawberry, the limitation is, more or less, present. Thus, development of a rapid, efficient, and stable transformation system is required for strawberry research and breeding.ResultsHere, using diploid Hawaii-4 (Fragaria vesca) seeds and a reporter gene of CHLH (the H subunit of magnesium chelatase magnesium chelatase) , we first develop a new, rapid, efficient, and stable transgenic system by the Agrobacterium-mediated seed transformation to silence the reporter gene, obtaining a transformation frequency with 10 % through a series of optimization conditions, including full imbibition and initial germination, shaking infection for 24 h, dark cultivation on MS medium for 3 d at 24 ℃, light culture on MS-Tim medium for 1 week at 24 ℃, and vector construction carried fluorescence label. Taken together, radicle-emergence germination seeds, appropriate Agrobacterium concentration and infection time are critical for successful transformation, obtaining transgenic kanamycin-resistant seedlings within 1 month and T2 generation transgenic plants within 4 months. ConclusionsWe first have successfully established Agrobacterium-mediated transformation of germinating seeds (AMTGS) in diploid strawberry (F. vesca), providing a useful tool for studying non-climacteric fruit ripening and strawberry breeding.